CA2463716A1 - Rapid extraction of rna from cells and tissues - Google Patents

Abstract

A method of extracting RNA from biological systems (cells, cell fragments, organelles, tissues, organs, or organisms) is presented in which a solution containing RNA
is contacted with a substrate to which RNA can bind. RNA is withdrawn from the substrate by applying negative pressure. No centrifugation step exceeds thirty seconds.
Preferably, the RNA is diluted prior to filtration and one or more wash steps can be used to remove interferents. In one such method, except for DNA shearing and drying steps, centrifugation is not undertaken during the extraction step.

Description

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: ' ~ : . RAPID ECT1~4N t~F ETA FRaIVI G~LLY.S AND
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~:v.,._,. , ~' ~ ; ';canker &~rid,c~tl~r diseases are rapidly being discove:rod:
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: :method of RNA extractiatf. from; cells, #issties,,avd other lbiQlogfcal'~;o~p~nenta.
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.moleaulartnarkers, amplifi~ble IiNA from sells aiid'fiissues uxust b~ extracted aad ' , , . , . .
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' of the invention at'e urganolTes, cells,-ccll $~g~enrts, tissuo, .
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The ~lufiions that contain R1~TA of ivtsrest fan. beobtaix~d ' ~ .
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~any'iuxd~a~mit izl:cfr' mass from the bxalogical systcan. , , . ,.
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1'~ , w ' ~i v ~ r . , . . .. 25 . , ,'. eadogeuotaa .ribonua~ase (RNAsse) stud removal of oontami.natiug;
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'Removing ctlls without RNA of iaterest ag,'thea~'anc~.
' i~ preferably dope by lys~ .
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' . s ~ ~aying tisa lysate from the solution. When' . .
'' woilQng with wha~e, blood; it is the red i . . . . -. . ~

: ~ oella prcacat witvaut RNA of iaterest ~siace they' ~ . ~, ~ . 5 . ~ ~ : blood oells.~'t make up tire .bulls' ~
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. . . ,. aontahait~g amamfl'nium~chtoride-~vtassiuin, . '. or. arntrtoaiur~i ox~,at~' , ~.oi~g aiaay c~tb~rs''~
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4 Fci tissues, oommeraisi RNA extraction kits such as those o~reci . ,; , ~ ' 1~ buffets s~re'usod. Preferably, these rGT~ ~aolixtion :arid:
~ ~'4 .1 ~ caataia's guranidi~,-t'hicicyarite . ' . ~ - '.
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_r , , .' . . , ' . ; :s't~n~ redu'cii~g ~~, _ , one or more surfactants.stich as sodium , , ,. ' ': , ' dodecylsu'lfate as~'ivell as ~

~'~ . ., 8ge.11t ~U.Ch. X19. ~-i'l3Qi'Ca15t001 J3$9. ~A8&e8'. ;
Q'lj'r'' ' ' ~ , t0 ~it~bit O~ lnaCh'~r8'te ilb9nLlG~.~a ' ' ' ' . ; '.
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, , i ' :.. '. - ~ y ; has patticulariy, useful disruptive and.It~rA'l~a cf~'e:Jsinsuti 15 pr~coti~~t~pxap~iee: ~ . ~ ~

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. ' , RhTA to be released ; . ~~ combinaziod: acts to disrupt nucleoprotein~ - ~ ~ ~;
" c~mplea~es, allowing tie;

.'~ , tests. Dalutiarn o cell e~ixaet~ 'i ~~'~
i' , ~ : in'to solutiCNi ~d isolat~d free of n the~pr'esence'of ~
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. , . . .. ar,pr~teias ~o, ooour.' . . . ; ~~ Canctions of G'>,C causes selective . '. ,.'; . y .. .
; ~ecipitation of tillu~

:~~ ; . ,. ~ ,: while RNAxemains iu sdutiota.
MbatpTeferably;'ItLTetimce'~c'~rot~.to eg', :; : ' buffer (sorn ' '.. .
; , . . ' ~ .:..iysis or liotrlogenizat~s~n buffex) ~ ;. . ,1- is used as it is, comzxiercislly avaiX~.ble ~ ~" ~a fGom'Qiageit'a~.' :, .
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. .' . , ' i ~ ~ ' ; . a colt of their RNA extreatitm fats.
.,~ ~ Lysis and, re~mowal of Gills aad,uu~ideeir~ilo . ~ .. ~ . . ~ . . ~ , . ~
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' ~ ; , , v, c~llulgr cvmpamontg can ba sided by ' ~ ~' mechanical disi~p~.on a :with a homogoniza~r,.l~Q
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:; ~ 'Tfiea'~ford, a sud~ei~tly large sample ' ~ '. slioulcl be obt'~inad so~tbat.
. 1 ~ ~ ' ' ' ' ' ' othvi tissues, .

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. , : :' motastsses'wil7.'t~ot be,mxse~d 'G~e ~ appmaah to sa~npl'~ag issuea2~~the'px'esa~nt ,~ ~thod.is ~ ~ ;

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. . , ~
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' _ ' ' v ';:'y:_.. , , . . . -~.. ~: , ~ :'. ~ ~ , ' '. ' ' w .
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;'f , ;:- .
. . . ~ ~ , . . . . . . . , . . , , .
. . y, .:

. ' - Y ~ ' ' ' ,.
, ~

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.
I

- , , ' . . , . . , ' .

~.-u.....-.
'y" . . ' , ' . : . - -.
i'' ' R ~
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, ~
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, 1 , y .
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-''.;, ;~", ' ''~. , ' . ' ' ' ~ , , . , ~ - __ ' ,., ' ;
.
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' :

.F . ' . . ' ' ~_ , ~ ' : p .

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' - . ~ ' ' .
i 1:

.
. -' . . . ' ~ , .. .. , ,~
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;

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. V . . ' 7 .. . . . . . . - i , ~a 1'1.'.! ~
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;, . .
~ . ' ~:- L~lteyNo;:CDS~5ao8 ' ~
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.
I .
.
, .
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.
~

:, ~, , ~ .
,,i.~ ' ' . . ; '-ta homo~.~c a laige, tissue sample, and evibsequ~tly~
pearfc~m : a dilution of the wel l ' ; , ;-.

:;
~.
-r , . . . , , ' ' ' y t tileoular"ter~txn ' . ' ; . . . ' -, to be used in subsequdn m g -, v mixed l~iogeniaed sample ' ' -.
.r , .. ;
". . , . - : : I' . , :.. .
,,: , , .

~ ~ .
,: .. ; . . . . .
; . , . , : . : : .
,,..~.. . .
, ~~

. ~ .. , ': addition.of ~tl~e homogenate td the ' 'A .dilutiva stepa~er homogenization and priar to the .
' ''~..'.~i~' ~ . ~ ;:
:~ ' . 5 ,:~RN"A' bindixvg step (aa descxlbod. bolow) is particularly advautagea~s, Tl:e dilution, slap' . ~' ~ ~
~
' ;

,',.- ' -- ~ ,' , ' ~ , '' i,~:. involvgs adding a dilueat that i;s a re~ge~t .that dilutes'the tissue , ~o~mogeaata without ;
~',~;
.
' .
' --.

.
.
. ' '?: contriibutiag roo RICA instability. Pr~efersbly, it i~ a rea~x auah'ae.;the tZL? 1' - , ,' ;
' ' .'i:
' : . , , , ~," , . , ., ~. - . .
a ' cn'bod above. Preferably, tha hamogeoate is diluted at.betw0en . 8 ~d 2 mg of ' . . ' , .
.
:
.

.' , ' , ' , -' , ' '~. homogenatc'pcr ml of diluetnt. l~o~e Prefer~b~y, the hope ig i~itti at bctwcea ~ , .

.
' ' ~, gbout 4 and ~ mg./m1. Ivios't pxef~eraL~ly, the hotnognnate is , diluted~~at sbou~4 mglml. ' , . ', , 10 ~ .

L:.;;:
. ' , y ' ' . . ~ , . . . ' , ~ . . . , Q . . -., . ' . .
'S.. ' .i - .
r .
, ' ~' , ~.~
.
~
a .
,'r''~,:

. - , - .
~~~ .
: . .. . ...
. ~ S'~c'li a dilution std aYlows for fast filtering, ae for dxamgle, thrau.~~h a'spiin,oolwan. 'It.' ~~
. , :
~

.~.; _ : , i..: . - .- : . : . . .
~. . . :eliminates ar ,g~atty red.'ttoes ologglng in spin col'uxnas, :. elfnitea t'~e heed for ' .
;
.
.
.i :
' ' '~.
~
' :

~

'. - . :s,~'hse V volumes ofho~o~enization hui~r, ell ~inates the uoedfor subsequent :: uanx largo ,, -',.
~
.
~
.,.

.
:' q :~ x S ' ~ .1 ~~,oe~.lfifugaxion steps, and can elWe'te the seed fore '' passage of tfie ~am~la through a ' , .
. I
.
~
.
' ~
, :
-' "
' '' .
':' ~;
"~ , . ,; sh~ddirig oqlumn ar device. 'Ultimately, such a dilution : sfep s ItNA extracrion :~ ;
~ -~ ' :.., ~-'v ' ' y, . . , . , ' ~ , . , .' . .
: .
. ' ~ . , , ~ ~~ :easiex slid res~ulta,ixtt a more etandardi~Od piotoca'1. ~
.~r . . . . . .
-' ' ' ';;_' -.
.
, ~
, ' ~
' ;i . , , . . .
. ; .
. - , ' . . ' ' ' .. ' ~ - . ,..
~.': .
'C ' . ' ' ~
.
' -,i. .
. .
. ' , ~;~;~.non ~'.A. ruaterials arc gamerally ~.u~dved.thrtiugh~one 1 or more wash ~ . , ::,,,i,,, ..
i'..,, , .:
;:' ;

' ~0 'vat~pa, aad~th~ purified BNA is thed extracted as, for examples' . by el~tiQt1 from a ; ; .
' a'~'''~
~' ~,' ' "
.
~~.
' ~~ ~ ' ' ;substrate-~onta~in~' cQluma; ; . . . . i . ' . , ~ ' ~
-~ . ' . : .
'v:' ~
~
:i ~
~
:
' ":~
'' , :

; ' v ' ~

' ' ' ' ~.
-;a .
' , ' , ' ' .
i , .
.I ' ' .
. ~ , .

" 1 :
~

.
' ' ~racipitated oiit of e3olutiva ' 'with'etl~aaaI'suii boW dtpo~a ' .
~ ;The 'RNA is sa2octively - , . _ _ , , ~ ~ ~
..,. ';fib=anolsubetrata'such' as .: the iliea sui~aoo ref the ~laes f'xbors,,'T>~e biudi~ of RNA to ;/ ~ . ' . " .
:
;
' - .
' .
~ ' ..
~ .
~ , 25 f , "the substrate ~c~rs isQidiy due ,to the diszutption of,the ~ water niole~ules' by the .
.
'-, .
. , ., ' :.- . - =e-salt, ~hvs favoring abs~ri~tia~ o'f nucleic acids tythe sylica.
:5 The bauad total . , ;; ' ' _ : .;
-- .
_: ' : ~
.

. .
.
.
' ,, . . . .
, ;:, .
,;RNA. is f~prurified'from ~atsmiaactiog salts, prateine'aud;cellu~lar impurities by .: ' ' 'iv , - ; , . ,.. . , , I .. . . . . , . .
,~ ' ~ ., ; ' . ' , . , ' v' ' ~' ' ' ;
' ' , ' $ . . ' , .
' .
- :I
. ' , .' , " ..

, , -, -.. , . . , . , . i , .,. , " , . i : ' . . .
. . ' , .1-' r w;

. . .
. . , . ..
, .
,':a. ' . . . , . , , , . '- - .I- , . ; " : " .., ~

. ' . ~ , ~ - :. , v. - , , - . ' . I , . .. .,. ' , . - , . , ,.

,.:.~ , -''., ,:. , , , .,,, -- ,, . . . , .
':
' ., ~

.;
., , .
a.' i.'l ..,.

's'.' ; . . ' :v..~,~; .
:. t ..

r . .
'6; .v~.,f' 1 .- , , . . . , 1. .~' ' , ' .4 ~y , . . . .u . , . 'r . , ~ '' ' ' ~
.
' ~ .:i ;
.
- i ' , ' ' ' . ,' .
' ~' ' .. .
~'~' . 1 ;
. , ' . ;
' -, , .
, I

' . .,. . . . . ~ .i - ' . _.
' ' ' 1r ' , , ~ .
. ~ - , r ' . of . ~ ' ' n ~ ., - !
f; , ~. ' '., i l . , .. .
. I . , , . , ~ .
' .i . . . . .
'. I'.,'l~ai r~i~.~~~~ . -~

f , , . , : , , . . , , . ' . , , J . -S
r , , , . s. . ' ' ' ~ the ttrt~J F.NA is e~lubed fi'oanthe me~bim~e ~by . ' tl~ ' ' .. y ' ~ ' ' .
' ~ siv~ple washing etej~s: Finally, ,' ' , ', ~'. . ~' ~ ' .: addition t~f nncle~se~free vPatar. ~ ~ ~ . . ' . i ~
'': . ~ ~ ' ' ' .
v . .
'~~ .
' .
i 'I .
.
' ' . . . .
.... y . .
' .. .
. - , . . . .
. , 1 . / ,' 1, . . . . .. . .
. 1.. . ;. . ' ' ..u, i " , '. .n .
.
' ' i- .
~, . , , , . :
' . . ~ , . ..
;; Several lots fQr RNA axtraotaon from tissues, based an "xgui,colpmtt"
pu~f canon have .

' ' ,.;
', " '.r ''~.. ~' ofthie ~n'ven'tion,''T.tle~ki't~
~ i~eenao~ni~cialized. Allarecampat~'ble'v~riththe-~,etb.od .,. 1 . ~
, .
'~ include those sold'by Invitro~a Carp. (Carls'had,: CAS, Strata~ene ~a ~rolla,1CA); ' . ~ . r~ , . : .

' ; L - ' $D1'~ioeQianods Claritoch (I''alo, Alto, CA), Sigma.-Atd~iCh ;~'~', (St;,Louis, ~1VI0);Ambioai '' , Tuc. (A,ustin~ TX), sad ~"rornaga Core. (Madison, Wlj. Typ3o~r11~, , ~hese1~'canmin a ~ ~ ~ ' ~;~' i ' r ~;~
.
~

' ' . ..-.
f::' . .
~
:j .
, r ' ~ . . . . 1 ' r lysis~,bindiag buffer havi~ag s guanidinium salt (4:4M c~' gieater)' itk buffer vKSah' . , ', ' . . -r~~ ;.'.

.. . 10 : ~. solutions having different dalutiane afatllanol in bufFer.ei~ei,ia'~e pzesancc or.ab~encc ~ ' ' ' ..
_ .
~. ~' of a chaat~rapid eal~ euah ors gaanidiri~um, and as elution 'bt~er, us~all~ ~.icZease~fzde ~ . ' ' ' .. . ' y . , ' v .- , ~ disttllod..watei'. Typically, contain .resg~nts such as; ' j ,. . . ' . , ~ . . ~ . ' . , ' , ' 17~ . ' . ' . . '. ' , . , _~ ' -i : ~ ; . . . . . . . . . 1 ;' ' . . , . . .
t -'' . :
: ~,r;-.~..
'"

' ' . . ' ' . .
Ly9is,Bir~ ~dix~8 buffer (4.~M guariidiriium-HCI, lflomM sodium: p$aspha~be)t ', ~

'r . .
''a .'' Wa$h buffor I (37!o ethanol,in STvI gt~suiduie-I3CL, ~~iTris: I~CL~, ~ ' . ' ' :.15 r t ~ _ . ~ ~ ' . ' ' '" ~f~te~. ~liuger II (~0'% i~tb~e~ol iri 2l NaCI, 2~lVt 3 Tris-H'Cl), . ' ~' . ' ' ~. ' , '.
:
.
i~
~~

~~~~ ~~~9 ~ ' . . , , ' r .7~~ ' n ' ..
' ''; t.- ' . ~ - - , ~N'caleasefi~ee ~crilc double distilled v~ater. ' ' : ~ ~ . ' ; .. ,' ' , ' '.
i' ,' , .
i :.~

' , ' ' ~ ' , ', .- ' ., . ., - . .

1 ;y. .
: I, .. . . : . : . . .. - . . :.. ,j .. ' . . . . .r.
~~ ~ ~ ' The use o~ a substrate to which ItNA 'binds or to vvhiclx a mates~$l .xs bott Ft~CTA ~

" .

tsinds to is what distingaishas this mgthad as a membrane based R1~TA
extraction ~ ~ . ' .' .;rs' ' me~ pmv~ded'ti~t . r , . , '; ~' methad:.'~T~, bul'ds to such material lay ~Y cbeuuGal ar phy$Laal , ,;, ;

, ~
. : ~ ~ 'tea ANA is':Basily,ralta~d bylor from the ztsaal, as for,exampld,, " . by aiutioxt with a ' v . , . .
; ~
: ' w ~

,; . , . , , , ~,' .
; , . . .
. ' ' ~ti7rtabl~ x'4ag'E~'t. Nutll~t~PtlS S'u~7atzateB 8ie iMlw early ti84d~lJi'~I7,em~1'~iB=b8S8d '~ . ' ' ' ' ~' ' ~

C_. ' , .: .: .. . ; .
C'.t' . , . ; . ~
'. ~ , ., ' ' , : : , .
-~ ' : ~ ' :,,' ' , '2~~; - mothodsJ IVtacherey-Nagcl siLisg'gel membrane'technalog~c~ie~on~
- siuo~.~riia~od and is ~ ' ' ~ , , "

=. desa~ribed itr 8P 049~S2Z wlticli is iacvrpoxated,her~ byx~efei~enwe.
F,saentlally,1~.N'pi, "v . '' -~., _ ' i ~ .
''j High. oomeentr~iio~ of c1u : v ' ' xs arisQZbeci' to a silica-$~1 membrane ~trop~lC RBlts ate ' , ' .
. ~ _ .
' r ':. - . . .- . ; , - , i'~ ~ , .i : .
:~~: . , ~ ' . , . , .i . ~ I
~ 6' v ~ .
I
~
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. ' . . ' ' '. , , ; , , . .' . ~, .

.
.
' ;

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1 ~ . . , 1. . , . , I ., ;
.
' ~. . ~ . ' 1 , ~ , ' ' , .

J.~~ ~ ' , . . . ' . . . .
I,'. : ' ' , . . ' ' . . . .
.~; ,,P
' .
1~~ f ~..
, . .' , ,. . . , , ; : ' ,' , , . ' ~ ~. . ' ' ; . - ~, , ~
i;,. .; ': ' ., ;
. ,.
.

;'~nYs,.(;:~: . n s . '. , ; ' } " , . .
' , ' . , ', i ~

. . . . ~ . ~ , .
. , , .
. . i , '!'. , ,.....J ... ~ ',.., .

' ~ , ~I
' ' . . 1 ~ , v .:
~
. .

t ~ ~ ~ . . . . ~ , , - ..
' ' . ..
. "
.

~~~~~ ~''i,;
-~ ~. i~aax~~r~:~cns-soot ' . . ~ .
.. . : .
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.
. , . ' ' .
. .
' ~ ~
' . ' .
~' ' ~ ' . , ~~ . ' "
~~ .
.
.,...
, ;.
~. iricltid~d is #l~e reagents ~iv~h'remove water tom l~ydaslted molect~l~a iu~aotuti6u;
. ' ' . .:

. , : '. :,polysadolisrides ' and ~oteins . da nor adsorb aad ar~ rem~tived.
~ Aa, wash step page ., .. ~ ' ', ~ , .
;A.; .; : . . . ' .
~ , . ., ~
, . . . .
' RNA is eiut~
ttridex low-aal~
ioonditians, . _ ' . ' ' . . ' , '' x v ~ .
~ ~ ~
.~ ' ' 'f; .' . . . .
':.: .r'~' ~' , .
, _ ' .
' ,. , ~ ': ' ~ , , ' , ' ~
~
.

' '.~ 1 5 : .
~ ' ~ , ' : ,. ,.
~ ~ ~4 ~;', ~usad as sub~fraxe$
.' for RNA e~stcdction ui Ion exchange resins are'also $equentty ' ' , .
~ ' far example, . . . lies a serciall;.yavailable . ~ ' ~iion . ~
' ' . .
;~aembzan~used systems.
t4iag~n .. :
; , ' , ' ~: ; :.' .~xchatige ~ ' s'esin,~ix~
~' wlaie3l a Y low salt : oonoent~tioa . is employed arhe,~ahe worlang'eample ' ' . . .
' . , : ; ' . , , ~. ,;.
' . .
h'lgh salt codcentratioin is ug~d du~ia~
Waahidg of .
a.
(binding) osed 'to' the suhatxate ~a ex .
"; ',. , .~, . , i. . p , .
. ' ~e'RN'A, ., axtd precipitation .', is visa an.alcolioL
_~'.. The ress'a.ia ' eilioa~ba,abd vsrith a hig3x ' ' v"
. .
.

..;~..:: .. : . ' ,,., , ' . '_. .: , . ' , ,. ,, ., ~ 10 , ' . , _'.
. ; ~ ~demsity . of clietby3atninoethyl ' gzo'ups (DEAF), ' halt conceattatio_n ~ add pIi are coatralled , .. , ' .y~: , . ,. - . .
. : , , ' , , ' w: t . ~. "
' 1 4 . ' .
.,, ~ , . .
., , .
. .~,y 'd"e addition aad vsc of thc.buffers .for u!$e with the aubatrate.
, ?A~ example of such ;
, ' .
. . . ' .
' .~' , 1 : : ~ , ~e . , .
;.. .
i' .
~' , : : ~ .
~' . .
.
' ant ion exchaage rssiri available from Qiagsa is as follows:

., . .
;. ' , . , .. . .
.''. : . , , - '.' , , , . ' , . , :
~
' i:
v ' , , ~
. . .
' ' ' ~
!
' .1 , , , .
..
, ' ~1'~i .
.

' .J. . , ,j, . , ' . .
, . ' S
: ' , .
. , ' .

. , ; ' I . .
' .

, . . i , . ' " .
' . 1 ' ' . ' .

' i . ; . . . , r ~ ~
, , "~ ~
_ ; n .

i' ' ' , . , . ~ ' ..
. , '!i - S _ ' .
. .
o ., ' i:
.9..', _~.i..
, ' ~ ' I
~
' ~ , . , i . . , . ' , s .;
.
.

. . . ' , .
..
,,.' .
, ,' .

'; . . .
! . . ,. , ' , . , . .
s , t [.
s .?' . ' , :,~ 1 .
' A

- . ' , , ' .
;.. ~J; ' . , _ .Y . ' j :
, ' .
. . , , ' .
; ' , ,i .' . . , ' ' 'a .
:;: ~ , : . , ' ~' . ' , i '"!'he l~o~t ' preferted , substrate ' ~, i5 a situple ' silica gel v: w fblla~uva:
~ .
' ~

. , '~' . .
;', . , . , .
. , , . , ~
.i , ' . ;
,i'3I, . , ' , ~~
' .
~
I

.

.

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, . .' , . :. . ;.
.
( ~~~
~

. . ~

, , , ~ .. ' ' .
. . ' ' ~ ' i ; , , . . . ; . . , , p~

''' , -l ~ ' - I . , ' , . . , ..,.i. ' ~
~ ' ~ , ~~-~
~

;.. .
't ' ",' ' ~
. .
". , .~ .
: .
;
. :r~
~~

, , , 1:, . , ,. , ~ .
, . , ,. , ; _ . , ;. . ' .' , , ~;, ~ . , 7 ,..
,~ . , , , ~
, . . . , , ~_r'. .
' x;

'. ' ~ . . ' ' .
... . . .
~' ' , ' ' ., _ . . ~

' ~ ' ~ .
y: , . . 1 . , i . . .
f. , . i . , . . ' , ; :
'. . ,i , .
:,! ; .
I. _. , -' ., . ' '. ,' ., .'. .'~, ._ '.
' .' .'' ' , ' . 11 ~ ~ . ~ ~ ~ v ' ' ' ~ ' ~ ~ ' ~' ~ "' ' ' r . , . . ' ~ ' . . . , .. ~ , l . ~ . ' . a ...
p .! ~ ~ ' . ~ . ~ ,~ .. . ' . . . ' . , ' . ,-'~~~. ...~~'. ' , ' .. . .
', . . . , ' .1 . .
1..!~.~1 ~ '..<'. . ~ , . - ~ - . ' . ' , ~ , .. ~ '. . . , . . . , ' .'''_.'- ..,~,'_..
' v .a.; .~ , . . . , . . . . . . .
. '" , : ; , . ' ~ ~'~ the presence'Q~'b~n~ buff~a, aucleia ~Cids adsorb to.
the,eilioa g~.. Sn the. ' . ' . . .
_~,: _,.a:: y . : ~ :., ~ 'r ~ ' W f~ireti-embodimcats ofthis iavoati tba substrate i~ ~ o ~, ' ~ is 'a v ~ ~. 'suc$ ~as.~ 1'': ~ ~ , ~.
'~ pre ~ G .
.~ . . ~' . , ~ . ~ : cc~I'umn. Mast ,, rs61 ; these colui7~s ate'cammerciall ob'taia ~.e~ g'as tlta ~ ' ' ' , .' ' ' , . , :;.tw . P . 3! . Y , .
:' , . .,~ . ,~..~' ~~ dol ~. ~ ~,ble fcoiu f~iogen, C+b~. : ~ ' ~ ~ 'F ~ ~ ~' ,' ~ . . : - ' eas uazas avai ,, ~ , .
't',s.:~~ ~ ' v . ' ' 9'1 ~. .~ ~', ~ ~. ' . ' . . -_ ' ' . ~ ' ~ . ~. . ; ~ .
.~ ,.;, ~,'; "' ~
' ~ . . ~., :,I~e~gref~rieci method for extracting ate pu~.rifyvag RNA. from ty.ssu~ :ssm xee.IesB'th~a ~; . . ~~ . .
P
. ' .. : ~Omg~app~lyiug the ~neagents coata~od is too RNeasy Mint Tell avai~abl~_ fpm. ' i. ~ ~:: . , ' . ' ~ i ~~~r' , , ..
., ~ . . . .. ~ ';;G'bH to the methods described herein, For extc~ctiem of iar~er'amQUata of fttNA fnoiy .
~t 4. ~ ' ~ - ~ ~.~ ~ . . ' ' ~ , '~ : . .. . ~ ~
' ' ~ .: f ' : . ' ' ' ~,lax'gar tissue s~m~les, the method desoribed below aan be'readily sed bygone off:'' . . ,.~ ; . ' . ..
iQ~
,'M'yskiliusin~among'otherrca~entoonfl~ra~ion$,'k~ts.oam~a~iallyavailabls,as ~
,',. ' .
' . ' ~'. ~ ' . ~ '' ,Q~A~3EN RNes~y MIDI or QTAGF,1'1 F,Naasy MAST hats from 4ZI~,f3~EN,'C~b'Fx. ~ ' ' .' ~'~~' :. '. ,. "''; M~, ~;; . ~_ , :. , _ . , ; ~'~ ' ' ~ ~ . ' ' . ~ ~ , .
..' .. ', . . : ' ... .
:: .. . . : . ~ ; ~ri using iguch kits,.the preferr~d m~er3~d according to the aaetsni .biventioa is ap .:_ - , .' , ' ' , ' . . . .
,, .. A _ , . . . ,. . . ~ . . , : ' .
~~'~'~~~'r _ :~ '~,~5 . ~ ! ~ , ~ ~ . , . ' . . . - , .- ~ ,~" . ' y .. ".
1.S .. ~~ ., For.s'katfing',tis3ueB Of less than 20mg, 350 to.lS~Ott1 ofHomd ' ~.,, 'on.Bl~f4~'r~ ',' ,~.' .~ ' ~1. . ' ". . . ,: (P~ferably, RLT badger. obtained in the kit from C2ian) igyac~d',~~' .the tissue, an'f ~,:' , ' ., ~ .. .' .
. . . . ~ . ~ for zfl:34ing of#issue, 600u1 of BufFer is.preferabiy added The ~mdrt~ataf' ~~ , .. ' ' ~' ~ ~ : ' .,. ' , . ~.' . . ~ ~ ~, = '' .' ,'. : ,' ~ ' '" .
. , ~., .~, .. , . . . ~omQgeaizatioxi buffer caa bc. scaled dependin~~upor<.the tissu~ sample; fiissu~ br.: ..' .,: ,: ~- ...
. ' . '~' . f . . 1.: . .' 20 ' sells ~aro'then di~vp~ by oae of the fa~lowiag wads ~2iu~g but not liriaated to .. ~ ~ ~ _' : .
' .. . . , '- : : rvt~ar: s~at~t:'llomogenizecs, bead mixers, m~or~ar sad posd.c,'and by h'ss;d griaders. . ., . _ ~. , .
_ . .: , ,. : ~. ~~ ",' ' . ' ' ~ Iiamo~rnizax~oa is usu~tly done by the use of a'disp:osable iissu~:gri~der (VCR ;. . , . , ' ;~ : ~ :. w . ' . . . , ., , ; . . . , . . , , . . ;: r ~ ~'-~ ' ' ° Scientif~c;.cat,r5~04~126'ar 1570-I24) when.matrual.oell and~i~ssu~e,dia~tian is . . ' ' ' ' '., ,. ~ ~ . '' ~. imdextakcn.' Mare preferably, homogeiu2ation is cva~ucf.~~. tlu~ugh.'tkte.use of'a ; ' , . . , ,1. ~ ,.
.'~ ~ _: . _ . '. .~ ~:. '.: ~ mechani'csl~devioe made for this gurpose~such se tbo ~~R~~Siseuip ~oiaog~e~zation '~ . " ' .
_~' ~~ fit dally available from Omux International (Warrentpi~, "VAj. ~ . ~ ~.
.
. ,~,i'.-~~~: ., ~: ~.,,: ' , . ' ~ .. , ,. ' . . ' , ' . , ~, , .. ' , ~ -; ' . , . .. . .., ~ .;, iv:.~ . '~v~~~~ ~' : . , .. . ' . . , . , ' : ~ - , .~ - ~ . ; ; . , , . - ,' ', [ s: , y !1' , ' ~, 1 i , . , . . r I. ~i '~. . i ' f . . ~ . . . ~ . I ~ . ' n '~ ' ~ '~ ' ~ ~ , . . .
Y ~'. 1':. ~ ~ . ~ ' . . . ' ' ~ ,. ' ' ~ . ~ .
. , , . . . . ~ i ' .. ~ . , .

.i':.:$;. ' - .
:y " ;
-'i :
:
. ' ~

. , , , ~ . . . . ':i , " ' ~
, ~ ' _,fl.._.~. ' '~
..
~

'9 , '~I.
~

.. . . ' , ; . , , , .. . ' . , , ;'~,;-,' ' . . .
. ' '' . ' ' . . . ' , - . , . ' ' :a , , , . ' ' , ~ ..
' , , , ' , , , . ' 4 , ~ .
.' ' ~,'=;~;';..i:.,' ..
' ' .

!: . . . . , -~ . . .
~. 1 ' :. ' . , ' ' "
. . . '.
' ' v:~
1,-, . . , . . , . . . . Y ;
: E . . . . , . . , . ' , , ' .. ' ' :r . 1 .~ , , . ' . . -. . , ~ . , ' . , . ..
~. .' ~( .
i,' ~;

. ' 1.,.:..'.~.,",~'~':,I~'.':. . ' . . ~ i ~ ' ' . , ' , !. Homogenu~tion bme is typically about ly aa.soanas .tissue , one minute and pr~fera~
, .~
:
; ~
~
~~,'.
~
; :.
~
~' ~
' .
~

" .. , , ' , ; _ . , . , , , v' .
. ~i$ viaiblj~ h~oinogc~nixed. ~ ~ v , . .
.

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. v , . . , . ..
. ~
: ~ .
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, , . . .r . CS . .. _ : .
E ~

.

, ,~ .
,.= , - ~ ~ . . . _ .
. '' .. rt ~ be desirable, as with de3~se ttssua ' e~mpies, on derxase~th,s viscosi'tyr . of the' . ~ ~ ~
. .
' '.
' ~
~~

.
' ' .

. fe:' ,~ ,'' ~ ., ', . ,sarnp e, IVIoet,prefarably, this :,~:'v~ is done bythe.aclditiort.ofha~o 'tion~'er~t'o~
'; ~. ~
' .
, ,., .:
:
.

_ homogenate (8m~ hvmogenatelml bu,~i to , ~ Z.mglzpl).. It,can~al~a be,doae vvith.oae' ', ' ' v~'.".
r' '' ~
.
, ' ~' :
,..
~

. ,~, . ~
'' r iacre passes through a needle (preferably , about ~0 g,~age) ,fitted to a~ RN,A'a~e-v ' ' . ~ . ' ' .
' :
.
.' ' .
..
, a ~ ~.~
~ ;
' .

,. . , .. : , , . r _ , _ , , ~,r Or Wlt~t $ l~eV~.CI: BllCha$ ~ ~~.
,. Bldg' 'Wlui~.lleBd.Wil~ ~ ~ ~ '.
. ;
, ~f...,''S
~~
~'~-r , ' j.' .
~ I, ~
I'f .~

. .
,'';',.,~ ' _ ' , .' . - .' ' ' , :;
:,.
:: , , . ;~ .
; oentrcfugatiQn. . . ~
;h:;_, "
.
' ,v . . .
, . . i . . : . , '~
, .
I ~
'i' '~
~, ,1,'~1~
' n , _ - a , , . . , , . ~

'~ 1 voluime of 7~r'o ethaaol is preferably~te~ and mbced~by ' ,~' added txr the cleared ly~s . ' ' '., "
"~~, ' ".
' ' ' ~
' ~
~
:~' ' , ~ . , . . . 1' , ;.
~,-, ' . , ,.' 1 ' 1~~~~ '' . .
. . ~, .
, , ~

. . .
; : . _ . . .
; , ; .
,,: . , ; . . ;
~; . , , , , :
.
s , i v' , ' - . . .
' ' . . , , , . ' . . . .. , .. , - ~ : . ' , _ . _ .
:. 1 ;.
i,,;
.;,;~~',~
~~'' :~
, "'- t7p.to abotu 9t14u1 of the sample prepared'a vessel housiing'ti~
':~ thus far x~ a~rplied ta' ~ . ~ . . ;
l.' ' ~ ' ~
' ~
~~
~
.
' ' , ~ ,' ' ' . . . . . .
. ItN~ bindhzg matrix (preferably,'a~ RNeaey~ . '. , ' t5'' silica gel tuini col ; .
~ ~ fmm the Qiagen ~.

:;;.1:: klt) ~.td/or discarded 1~y filtration and p~'ferebly the .~
,~ has ~ppased~t~ ceatrifug8tioa~ . ' ;;
.:' ' ' ,__ .
.
' ' . .. , , , . , ~. . . - ;, . ~ . , .
~

';;' Preferably vacuum is applfed a't abaut,l~I'tr~,I3 pei (,~s,te ,x. appliestion ofva6uum.. Qie ~ ' ' ' .~v, ~
' .

, . ' ~~ge ft~~' ~ll,v~u~u'n $t~'ps) for ~ step$ in wh_ ioh' , sbQUf 15. to ~0 sec. Hare as ;ia_ ; ' ' . . "
: al .
, .
' .,~
..
.

1 . ' . . . ':
' . ~ vacuum is $pplixd, the time in which :
. it is applied ~ati'b~ ~ecrea .. ,, ' :
, ' ~ed .~ith aiY ~Crease . in vacuns~i, ptesau~e provided that it .
, , does not exceed the capecit .
' , ~~ ~ of ttte matrix to , ' .;s;
' ~
2p ' .
' , Wll',~18~i~ $~iGl1 pl'C$StltB. . " . , ' ~.
.
, ' 1 i :
~
~
' "

. _ . ; . , '. ., ' . .
.
' '~

m ,y ~ , ~ . ' . , . , .
, l . ;
a , ~
~. ~
'' _ '~i7$u~f~~r ~prefex'ebly about 70Q ~,1 .
~~ csf ltW1 'bu~cr frog the ~ ~' ' .:
:: l ~iagen fit) i9 ad~ted y . : . , : , . ' .
, ..

' :

" ': ao t'he vessel Housing the ItNA bindug ~,sy cciru~iu). and ''s'. ;matrix (paeferably, RN'd the' : . ,' ' ~
. ..
'~'' ..
.
:
' ' ,.

._ .' fiow~thio',u~ fluid us ~ovedland ar ferably tl~ugl~ ~ ' ,i-:' discard~od by filtration pre ,' . .. ' ,I
.,-.
.
: .;
, .
~' ~5 ' ~
1' I' . ~ltsatioa ~aud'pa'efersbly the application~'a ~Eube a~
them.
; .' of vacuum aad. colleati . ~ ' .
' , ' ' .

_ removtd' at~d!'aF Qiarearided. . ' _ ' . ' ' . , ' . , '' r. , '1 ;!
.
~
.
' ' s , ' . ' . 1 ' , . .. . ~ . .. '. , : ' .. . ' r ' g , ' J ' ,' ' '~

j;:v ". .: , , ~ ' .
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..

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~ .

~:.~ : : , ' ' ' ' ' . ' . ' '. : . ~ ' : . : ' : .: ,' ~
:~ , ''i y ' _.;
.~ :'.
, .

v , A second''i7Vash ?l:(pxeferably about rnm thp. ~.~it)y.. ~' ~OOui ofT~"E:b~ffer ,~ . ' '': . : ' ~ " ~,plpet6ad on''to the column. ~ tllbe,lB
''' CloBEd ~OII~~ ~lld~t~'ffb~ ~throu~h.ia ~ ' _ ' -' . . .
y ' . ; . -' ~movod' andlo~r di'scsrded~by filtration,ati4n of vacu.'tmi $nd pref~ratily the app :'?a~4 ~ : ' ~ ,.

. , ', ' . ' . ' .,' ' . ,. '! : . . . .~ ... ,' ' ' Optionally, v~ra~b. buffer (preferably.can,be added~aga~i about SOOu1 of RPE buffer .'''. . ' ' .
I

E . ~ ~ ' - to tb~e vessel o~e~
'~~. ' ~ (px~fezably; Rnea~ column). and the .
~ . Th~ tune=fs g~nfly'c~ ' .
. ~ - - ': ~~ ~ , .

, , .
. ; ..
,' ~ ' .
~ drl~.. 3'hovessel is preferably plaoee( a:~eW. ~ ml . '- , .
it , :..~., : '~ .
., .~ubstr&telmemb~ee ia , ' . , ' , ,' , , .. ' . - ' ' ~nlloetion tube and,CeritFifuged (p*efezably,ut S,~O~g~. ~'' .
. for Z'.a~ute at~abo ~ ~' ' ' . .
y , . .-.~ .' -'. ' -' ' : ' , , ' _ 10 J , ~ .. , ' . .;,;.,:
~ .~
' 1 ~

' . . v For ~lutii~n, the vassal. iit preferablyj ' tiansferred to a~nei~i ' ' l.~ml .ollbc'tion tn'be ~ sb, ~ ~ .' ' ' 'J, . ~ t . r .. ' n , ~ ~~~~ .
, , .~0~5~ base-frea avatar is pipetbed dit~octly;J
onto irhe suhstra .
, me~brane, ,T#ie' , , D ~lkt for about 3d . ' ~ ,~.be is cIosed'g~ady~ and eentrifuged.(preferably' . ' ,: " . ~ ~
:~~~:
at'aliout Ifl,00~ S
~

' ' . ' secand;e).to ~eh~to the RNA. 'If the RNA.:v yield is expe~e~d to e~ ~
~ ~eed.30t~ t'~e.'s~eps';
' .~ , ., ., ~
, ' : t f ~ , .
v ~ ~ of this paragr$p1i are preferably repeated . . . .
.y '15' duri~sg elt~tion,'~' . ! , ' ' , . ' '" .. , k f. , ' . ~ .I . . .- .
7 I ' ~
Y
' ~ . . .
.: . . . ' ~- .,: '.
, . . ' . . , j ~Saue (QIAG'1~N ' , ' The Rl~Ay pmtaeol fof isolation of total . . .
RNA from' azuma~

. v mc~ of at lca'~t b:iain:
' 1~NAeasy 'F~andbook, Juan 2p01) raqui~ . I y ~. ~ ' total aantbn ~' tt ~' , , , far the C~nt~'ug~'i's 43sr~c a~nii 8min.45 sec when accelel~utio~' 1~' ' ' and decelet'atian ti~me'i r . 2U ~ ~ii~alu~ed; ~a ooazpa'risoa, total, time.foratioa stops ~ thcs' " all flltxatian and ~centci ;. ' a I
, ,y: , . , ' . ' ~ , t . : . ' . w ', ' ' : " , . rapid m~tliod ofthe,instant iaver~tion ~la when ' . _ ' . ' ;. is 2min and 15sec; aztd 3ff ' , .
' ';
;
~
;
.

.;,I; . . . , ' , : ' . . , accelers~ic~i arid deaeleratiou of ..
= th$ centrifuge a=e iuoluded"~ vn.~ adsiitioa about :' .
! ' . .
' .

. ;. ; 30aGC to ;lmia, for tissue homogenization . and '~buut 4Saec ~td ~ ltiditi fpr ~amplc:
~ . ~ . . ; . , ' ; ' '; . ' ~ .
.'''' ' .~ uzanipulatiou (utcludin~ dilution and ~t mixiag~teps) ara poamio~ to t~'xnetbod ' oftlio~'.1 ~ . .

~: ' '~ ' . ' .
~ ~, v ~ ' ' - . .
. '25 , ~' ~ ~,ventia~n~anc~ the rne~od ofth~
.t ' piior . art.. .

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.

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'v I~ocl~at~~To, CDS-500$

;. ,.
' .'. . .
.,;:_:,~<.~.' , , . -' w ' . . , ' . . . ..y '. : ' -~' ~ ~. - ' ' ' '. . , ~r~ -,, v ' net"k~d of ~s inventitan is le'ss.f~aia ~ei~t minutes, . . ' i tire t ime f t ' :' 'Ib u , ~
o e t~
a t s, t .
Th ~ '' .

'si '. . .
' ' ~'.:~ ' , .'. " . , ~ - -~; ; ' ~1 ; ~'; . .
' ~ ' aa six minutos. A totat~ extzactiarl Limo of five xz~i~t~t~es is particularly , .
' Tege t'~
refeiably . .
' " , ; .
. , , , : .
p ~

.
:.'r' . .
p ' .'n "
in '. ' . ' . . ' ' $$8~71P. 817 8'~fil~l 4~ Wh .P~ '~E'r ~&~~8 1~ $'61af119 lYra~h:abde. ;
~

' , .
, - . - . . ' .
~ ~
' ' ' - . , a ' , : ',' .
~ '' ' .
' ' ' , . ' i .

i .i . , ,. .~'~ or .
:Ly ." ~ e'rat~ly . , a~iifold is'pr if . . . , . r" tlcl~ as a QL.4 vac 24 Vacuum 1Vt ' ': ~
'' ; -''';
; vaouum ma~iftild s A

, , , , , .
.,,; , ~~ ' and wash sciluti~ons ~ ., - roeoesing of spin ciih~,s-in'parallel. ~ Sample~a~.
. . ' t ~cuunx ; ~ - -~
~
de , p . .
. ' -n ' ..
' .
caaivet _ ' 'aie drawn t~ou~'th~ column membranes, bY ntrifugation,'' ' , vacuum xnatead. of ce J~

. ''-, ~btaatio . ' ' ' '',; . :'~, -' ' roviding gr~s~er ~peoii, and reduoedhands-ot1a andpu~r~oatioa of "'~' ~" time iz~ the , ~ "

, ;
. . ~ . .
. .
.

. , ...'. . 1 - . . , : , . ~ , .. , . , , , ~ ~ ' ; ' - . ';RNA ~nm oel~ and t~ixsues. . , ~.; . , , , . - ' , , .
''. ' ' ' . ' ' ' . . _ " ; ' ' -~' . ' . S
l':. ' ' . .

;;'. ~~' ~' .
; ~' . . ' . s ae-moll as'bactesia, ~~ ::'Y .-~',; :- . -. . v . :,Tf~is: mood can be used to extract RNA from pleat snd a~aZ cent ~':r' . ' ~ I asx;:plants; ,and Wema~us fiur~i with m~tbod:' ~' _, ., -~~~. an appropriate distiionf _ ' _ '_ .
. ' ,:v~ ~
: ;

:. . , _ . . . .
i: ;~. . .... ~ ., , : : .
x i: , ., '' , , .
.
' .
. -. ~ , y l . I , . a ..i . , , , .
: ' ' -; ' . , . . ' ~ .; ,' . ' . . , , ' ' .. .~ ' ~ ' ' ' .

~f.. ' . . ; , . 1 . . . ' _ .
,, ;~: ~ , , , y , , '. , - ' i ' -.~: .. . , .
; . ~' 15:':
.
' , .,. i . . -' -:. . ., .', .
.. . . . . , ~

..., ;'' , j , ' - . .
. '~';, ' '. ;~g~-tfme'pCl~
' ,~
. ' ' , , , , ; ~ ' ; 1 -;;Examples in ;tl~~ present invent~to~i : ' .~ ;::~ are based as i~xe u~ of'r~al-time p~.. ~Ia real~tirrio ', ' v ~ : .
. ' ~

. ~ ~' . i ~ real tip durlng~tbce' ' --, , : .p~R the products of''the pQlymerase chain~
! -' reactioix are ~tanit~d ~
~
-, '' : ' '' :~ponontial phase ~f PAR rather ftlan .
~ y ~ , by as end~poiut naaasvremen' ti. I3ence, 'the ~
' '" ~
, ,, .
: , ' .. .
, .
~ 11~ ~
- ~ ' ~

. '. '' " t ;
' .q~antificatio~, of ~3NA and RNA ie much more, : , ' , precise and'reprod ~cible, Fluore$eenic ZO
' ' ~ , ~roduct~a~plx&ed to 'v - 'values are recorded i~ri~g.every aYcle .
~ arid represent the amount'nf ' r ' t v~
~
' -'';, b egianyng ofv , . .
.
. '. yhat-point in fho a~mplifiea~ion reaC,kion.. .
- 'The =as~re tezrr,~~ates,prr~en tjat the ' ';
;', .~.
' ' .
;., -; r' ' ~ 1 'the reaotiau, tli~, fevv~ ~um'her of ~vhi~ tie - cycloa~ir tales to~reach a pflint ~in ' ~ ' ; ' ' ' ,.
; ' .
~' ' '' ~

''~ ~ . ' fluorescent signal is first recorded ~ as statisiaoally si~oai~t above 'background;
. wla~h. ::' ~, , .
~'" ' ' ~
, -' ' : : . . . . , ,i .
. . .~
~ .' 2f ' ~s t'~e definition of t'6.e'(C"t)~valuds,~
'' The concept of the threshold cycle (G't~
. aLlowo for ' ;-:
:

' . .T-f CR: . ' . ~ ~ . -v ~ ' ~ , ac~iaane and-u~ucfble quanti~ieatioa us;ag fluonece baked R

. .
. , ~
.

. ~ . , ' ', ' E#a~tage~eous aebectaoa of FCR products caii ' be liexFarmal usie:g ~any dtfferei~fi j'; ' .
'':
;

. , , , . :i' -.:
-' ' .

, ..
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, ;, ~
, .

, ,11 -, , ' , ' . 1' ' . . - . - ' _ ' r.
F . . . . . ' . , i. , i~.~, -, ' ' .'C

. .
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, .
' , . . . . . ' .
. , .

7 .
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:

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' , ' ., : , ., ., .

y' ;~ . , ~ 'l :
':. . , , ' . ' . ' ,' .~
. , . .
.~r .'.~~aclcet No:.Ci7S-5008 : , , _ '-~ ' ' ~

. .
' ;:. ~ , .~ , i . ' ' , . . .. . , . , , . o., ~;~oa m~tods ~nCludixtg base babcd on double-:' str~c'~d T~~IA ; , . , .. ,;
. bmdiaog djres.(e.~
, . . ' ;;
. , 1 r, ' .?

.' ~ ' ,'.~YSR Gi'ee~); fluoragenec probes .(e,g.,,, 'a , TaQ Man probes, lV~ole. ' .
~ . ' ' , , . ' = ' , .
. ' i Beado~s), sad:
=' ' ',..
.

. ' , _ .. ' ~di~~t Iaheloct' primes te.~. Amplifluor , , ~, , ' , ~zimere). ~ , . . . .. . , ' .
~ ~ .' ' , .
v, .

' . . _ '_ .
; .
' : ' .

' . . , .
' . ,, . , ,~ 5 . ' i',, . '. ~ . . y ' , .
,' ~ ';"Ex~naple 1.''Short Cea~rifugatfari Tlute ~ ~ ~ , ~
~
.
' ~

_ ' .
~ - ' . O breast lyaip~t.aodes were purchased rom'Ast~and.
; :r :xuc: (ITetroit ~ , _ ' ' . ~Tn this 'exampl.C, tVY

. ~
's . . .
._ ' , 11 ~ ,.llril): . ;,. ,. , ,. . . . . '. .,, .~' ' , .. . , . .
:~., pa'rho~ogY (ALN~1~ ~ad.~ s~conc~ uode~
~ ~
. :-1 :, 1 ;~ ositiva '6 ~; ;O
~' med as aaae . r , ~ vas ., . fl ~~ l e i r ~
' , ' ., y e ~
oon r e n ot x ~
~
.

I' .~ ~ . ' '~~ ~ '' F ' Waces tiv~ fbr csr~r (,r~NNI;). ' ' , , . ' . ''' . ~ . , . ,, . , ' ~ - .
q ~., 1.' ~, :
' . ' r .

. . , , , , .
. , .
~ , "
., I i. . . ' . ' , . 'ia, ., : . ' v~ ~. . .
~ ~ . '' ' . ' ' .

, ' .
'; ~ . ~. ~ ' ', . ' ~ ~I vAs a control,. ~Qrag of tissue ~~o~a . ", each node wae'exiraated~.and p ~.uit;d ~ ~ usi~ the .

' ' ~ ' ~~IAGE~rT l~easy .Mid ~~'rooedure as . ~ i~eooinmonded'b~r the ~~xianu~g,at~d.:as' . . ~ . . . .
~
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' ' clescribecl e~'bove: Triplicate extractioas ~ from a sine. hoaiogeaata ac~es~ per~aifned ~ ,rn~
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' ' waah bu$'er; ' . . ' ..
' : ' ~ ',C2~$g~ea R~Teae~ M~i Procedure, except ' 15 tl~t a.Rer the 'iaal ;~eddutortx'rf RFB
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vvas ' '' ' Cx~man~~ and'~tlie total RNA yiehd vsrae' . , ', ' ' ,. .
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f'0 - , vg~.,~'Aase free avater:'Aseuming complete to'cDN',A~, aid,' a aoaversioia ofR'ht' ~ . . - ~ ' ' ~

':~ i ~': ' 'conveisi'on f~asor off' lyug equal to oa~ration of ' ' . 100;000 cell equivalents 4~),:a ' ~<' .' v -' , ' 1004CFJ~.~L' iw80mlv1 Trl~ buffer was used, a' for sIl'subgeque~ real-t . ~ ' .
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. . .~' ~ $B anti' ~~S IIt70S0~jlGalES Wittl ~11m,~'.RNA ~~.011., All'88~11~11$g hS~ S , .

"t . ,..' , i. ~ . , . ; _ .., . ~ . . . . v .' , .d: ~,losoi~ beak ratio near l.d .ar gc~sater, based cm the ~.gllemk ~ .. ' Bideziei~r data, al$o' ' . .
; . ', ' ' .~

:; a ,'_ ' . ... ' ' ,.
~ ~. . ,~,p, y~ 5 ~a~t5' RNA tom th0 slaarte~ed oontt'ifugation p ott~cal, The,tptal .
.
, ~
. .
' ' .
. ' .'~ '. , , ";' . ~ .
; , ' . . , t . ' , , i ~ , ~ ' . : , .
. .>, ~,e for i~I'~.A, cxt1'aCtion vas 5 min' 15 eeo: ' ' ~

. j ,Y. ' .
; ~ . .
.
. ' " , , , . . , , .
, I

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~ ' "' ~ ' , . ',': _ ., ie; .;~ . ~
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. .
. ' i ,,,w ~~'' ~

' ''1 . ,', . ' ...' - ..~
. . .. .
' ' , '. . '. . ' , ~
, . , . ~ Example.2. RNA.,yfeid, reverse tratleCript~an, sad rn~c Pd~t ~.mpll~c~~la~o. ~. ~ ' ..

, , ' , - . . . . ~ ' - ' ~ ' i - . , .. . ~ t ' , ~ : ,, "
' - . ..
' ~b ~
~
d ~.
tl d t l l ' ~ ' n ' ..-.
. c$iC
raast axi ary ri~a xla~a W
s expehment, a e w ua a uBOd. .~41 es , , rn ~1 t t l.x ' ; ,. ' ~ '','..vvera tha ~JAGEN RN~asy Tit ~ost'74~iBi, QIt~~,F, Iac.
' :. '' Y~'le~ia Co~d~ation). . . . .

' :'. : ~ The followix~g'suppliea and equipmont also were'pur~ha~edfzosn . j ;,.... t~I~.t", rB'N rNC; .' ' '- ~ ' ' -' ' ~ ' : ',' (I~IAvao 2~.~'tlrariag appa~'LUS (eat 194U3),'QIAsdex (vat .r: f 7~65~6),.Q~4vec . ' . : ; ~ , .
. t : ' ' , ' v ~ ' 1.

"
:~ , .~. .
~ ~- ' , .v' '' .
'' - ' ctantuectox,s (sat ~194U9),'anci YaoCormeotors (ost 1.9,4,f!?)' A1r ot'her;reagerite vvece ~ ~ ' ' . , -v ' ~,'. : . . .
v 1 S . - o~taiaed fFOm .~ourc~s 88' de~ribed in Ele I: . 1. . ~ ~
; .
-, , ' ;" : . ' ' ; . ~ , . , ' ~ ' . - , .' ' , ; . ' ' ~
' ~

:'~ i. ~ ' .:: for this,e~~rim~nt tissue ho'mogexdzsititin, 54CJmg,ofb~east,noile;~ea~e ~ ;. ryas ~suspe~isd ~ ' .
. ~s . ~. .
.' ' .

', . ~,, .'. is 1~.8 ml o~8ilffe~;Rrx and.TtQmogenized prior ta.ttse, Tha ' '. rasul~taat homos.Ee.Wa~3.'- . ' 1, . .
, .
. .; .
' i. ' 1. : . , . h. ~: ' .
1; ~ ~.~~ . . , .
'. , . ' , . .
. . y ,rrCS~(~'IZ! SeVe~. ~t 'xiay&; ' . .' , . . ' .: .'.. :J' '' :':,~.;'~~ 2p', - ~, ' . . , , . , , - . , ~ . . , , ~ ' - -' ' ., ' ' ; :
' '' .': , .
~ ~' ~
, r .... . .
i ~ ~ ' ;' P~. ~v~,luatioxl o~ Vaovium Maaifald. Th9.s, ~e~it was pe~fornned using a .: ~ y ' ' ' ',.

, . .
a ' , . . ' .
Q~V$0 24 1~ app With ~ sspplled. VSOUUm. , .

. _ ..

,~'I'~ ~"'.' ' . : . - . ' ., . . ' . . , t " , . .' . . .
~
' ~1 -.

' ' a~ , ' '~ .1.. 700~,L of homogenate were placed into' a t~IA.st~xedder.
~ -. ~~ ~' co'~i 'and ce~#ag~at ~ . , ' ~, ~~ ~
~ -' '' .
; .. .

' , ~. , '_ , ', ; . . , , : i . , . ~ .
; zs- ; : 1~''4,Qi~0 ~tl',lV.f ~o= 2 min.
' ' . :, , : ,, . .
. ~ -, ' ~ : 2. . .Aa 1' ~rolm~e of 70% ethanol was added'to the 1~'sate said ~
,m" ix~ci by,pipettiag. . ~ , . .
. .
~
. ~

t~ ; ' , . , . ~,' . ~- . , a . " y , ,:,, :, . ;
' . ', The ne~ct step wdB caut~ued ~vvitiLOUt delay ~ -. _, . , 'i.-~ '.. . ~ . . ~ . , , . , ~ ' ,!; , ' ' , . .
: . ~ ,.,:_..
, .
., .
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1 n ' ' . . , t , . : . , . ' .. ' . . . . , 7'; ' ''~ ..
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: , , _ .

y '' t ' ~ i . . . , . ' , , , .. . , 1' .

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.
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. ; :,~., . , _ 'a-,. , .
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; ,:'H'. , , ' ,~" '::lf , 5. , . . ; ~ . ' . ,~ ,~
w~s~..~,t ~..y ' ' , ~
t f i .. : .:,i.. . ' :~ "y ~ , ~ . . , , 5~,-' ,/
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. ' ;

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,' ~ . . , . . ;t:". .
1~'f1 , .
: i. , ". a ~ . " "':f. ' ' ' , , . , . i ' 7t~J~t~A a~~s~mple~~r~s applieils~tb an R~lB,asy~x~ini~.ed tw a,v~uum , ~
coiuma atta~ ;
.. WOK

' ~ , ' , .
..
, . . .

' n Y.h'S~. . ~ . , ~ y,a.~l ,Y ~.
~..s- , ~ .I , , ~ = ~- ' .. TT m ~y/. I
Jy VS.V. , ~
LLiWJJ..O1.L. YUA/44 W.VS Wn~~ ~~~~ ~ ,~~ ~~1 ,iV

s\ x , ' ' Y r. .. . t ~ .I,.~ ~

' ' . samj , , .
- preseuie .' e~' , 7i70~tI: o~Buffex RWf werre'-;~iplied~ va to ~ co~.uma, ~d ;the .,v ~, ; ~
.~~...~ ~_.~ .
.:> y ' ~ ~ ~
;s.: ~ , .~~ a~p~yf~='t~~ ~,a~n, v , ' ' . . ;
, .
, :
. , , ~~ , ' r ' ~ue vacuum pressure aet~ad aato'th~.aolurnn~lt'~a~s , ''f~i~ .
~~oa~. oBuf~er RP!E weie ~.~

, .
' , . .. , ~ , . . , ;~" , E.was added to,ahe';
';, .. .
' , ~ . . . . ., ., .
. ' was a~lpl~eid for t'he s~a~rie~dur~han.
. Another 500~1L, inf l~t~er F
' ' . , ' . ' . .~
' pi~ssiir~ applied fQx tire eama dti ~a opluma and the ~a~e i~acuur~ tion, .
.

, , , . . , . . . , f ~ . : _ , . . , , , . , , ' 14,400 ipm for ~1 .;.' -rhe~ calt~mu was .plaa~od iato a coll~ciiouy : . ~
:,. tub aivd aentxiFu~ed at -~ ~ ~ ~ ' , .~ G:.
~ r v ~
' <
-' ' . , , " , .
, . . . ' 1 ~ -, ~ .
~ ~
:,~
, -. ~~~,~.falusti.~a " .. ' . , . : ' j . ' . . , .

' et -fzee water; '. .' 25 ~RNAs j~L ~o b pi 1vt .
i .

~

. . e : ~ ..
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e. ~
p ta ~ new .
m ma was trans~errad The coh 1:0 :
' ' ' ,.,; ' v, . , .~ ' ' .
. . . , . .
. . ~ ~ dit~otly Qu#o 'fhe meanbFSUO, . . , .~ , , , . , . . .
.. ' ~' 'f ' ~~
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.
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:
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v '.

F, .
ij .i . . , , ..., .
v :i.
. ., ; . . , ' ' .
.
''S ~'I'he m~smbiana wss aonliifuged for b0 sec to alv~e: .' . ; , , ' . , y,.;~:: , . ' , , ., ' ' ' ' ' ; . . ' , .
_t.' . P . . , , ' ' . , . . . ; .. . . .
i .;.:,' "

' llii . , . _ . . :j , . . .i _ p , ' ' . , ~ v tt ,.d~
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' , . .
y.y, . - ' . ~
" , , ~ i .

~
, .. , , detarinine the ~i~cot, of x'edlucing the ceafifi~gatxon time. to 30 15 ~ . a~ p a~rfi Ix was performed to . , ' , , ' . .
~
. ~ ' ' see as moll as ahe a ae of a ~tacuuia manifold . , ' ' . :
' ;' ' ~
;
, ' ' A

. ' . ' , , , .
: .
..
. .
. , . .
, ' - i 'i ' ~ ' ;: ~ v:1. r70tii.r.L of haniogeriate Wem placed . iatc a. t~IAsln'odde~ oolusd, ~~ad,c~.fugad . at' ' I - - , ' , .
. , ' - ..
~:''' ~ ~ '~ ~ 14 ~ 000'R"1?M fdi 30sec, :
' ' ~ . ' , ;': ~ '~
:~-' v'.j ~' : . . ' - ' 'i;;
~' .'.

_ , . . , .
, , . . .
.

' ~,2, Aa~ equal volume cf'70fo ethanol was . added'to '.lysat~:and i~nixed by ptpettfag.
.
20.
~

' , -'. ' , ~e ~iext step was ~oantlau~d ~vithvut " delay ~ ' ' , ' . ~ ~ : . , .

. . ~ .. . . .
. t y, ~
, ~ ~ ' . .
. ~
. ' . ~
~

~

1 ' ' ,; ri , :
. d,ta a yacau atta' ' cot ~tNEas ie was lied do ~
~ 3,. ~OOiiL of.~amp pP ~ 3~ z~ ~ . - ' ' ' '' : ; . , . ' . ~. - .
., ~ v ~ ,manifold :Vacuum was applied at 12 psi for' 1,4 sec,, ~ .

, _- 4 . '~OO~,L of Ht~fFet RWT Was applied to tire column, and vacuum, applied as abo~.
. . , .,' ' :~'. u#~er RPE were pipetxed.o~ta the ~olu~nn ;'~ aaid'vaci~m appl~ed~as'abave , ; ~ ~'' ,. 5 ; ' SaO~tL'of8 i f ~
.
:

, .
:
. ~
' .

~~~ to t~ :colt~n.anc~ivawiimi applied ae ~ ' ,' , . Another ,5001tL of HufFcr RPM: were $pplied ~ ' , ~:
' , ,. ' . . - . . .~
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f ' f. . , ' . . . .. . ' . :
' . . . , ~ . .
aboVe .

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'~.5'. . ' . . . , y ~i , ' . L . .. ~ ' ..
, ' . .- i ~~ . . . . ~ - , . ' I .' :- ; d ~' :~ .. - ~ ,' ' : . .

. . , . . , .
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NOi , , I~S~Jr4O$ , ' . ' . . .
, t j . , , ' ' , - . ~ - ..
. ,, - ' ~,~~ 1. , - :l4,Q14~.~'pr~ fQr~' . ', . . ~ cod wee placed into ;~ oolleetiau -- ' .. ' ~ v tube a~rJ~d oesit~ri'.C'~g~d_ al .i f, - '~~ . ' : ' the colwma. , ' ' ' , ' - . , '- ' . ' ' r ~' ~ ~ 30sea , ' ,- ~ . ' , w. .

. ' ' ~ ~ ~,'~, 2~).tL.ofvt7.VAse~ .
~ ';j , , The cc~l a~dew l.5nol tube. five .r,~a~er vas ~''.
; ' . ~ ~~ wa~~tra~ferd to . .. ., . ~- .;,~~~g~ttG,d~ ~~Ctly'on~ T~1~ membr~rie. :
~ ,: - . ' ~ . ~ ' . . , ~ 1 : , ~ .' ' ~ '.

' , 1 . .
;, ! . ' : ~. lion, was Goaducted for 3,0 sec .
, s. ~~ ~'~I~u to elute. ' ' ' ' .
~ ' -" . . , . , ' . .
. .

.I.l., , ' ' , , ~ ,. ' i . ' .'' ' - .., ! ' .
- .

.-~. - ~ ~~rtll~.ofldv~~r apeedc~,tti. 1 ., .' ~ ' ' , - .
' .~ . ~ Use . ' _.' ' ' ' , ' ~ vh. t~e , ..F~ic;:~est, ' exp W .t, a ~uoaeZ ~loNtvss ~atrifug~,.p~~~rm'v' ',:
' , ' ' - y ~~ ~ tap 6 a~d.'7 of part TI'abo~e , . : ' ~ F at t0 000 r ;. . ; , : ; , . ester, as used in s s I, ' : a , ~' - ~ . '' ' '. , ., ~ ~ A ' .
'~ ' ~'~m. .;
. . , , , ,: , y;:~: : . - 1, . ' ';' .
;~, ; ~. ' y . , . .
;t, 'i~ ' - ' .' : ; ' .,; . , , r v.. . . ~'~ac'~, . .
' ~ ~'~'..F - ' , . ' ! . .. : .
z Colt~ti Arying step, ~ . , .
, . . . .

. .
' , - , '. ' ' .. ;.~'a,~g~vecqritz'oI, an. exgerltri,eut was , ! ~ , included~ia vYbich s~'g- l~t. IC1'w~s . .. ~
s ~ t ' .' ~ . ' .. .
' :
' ,4.. ',1~ .. . . , ' ' ,A ~ " 1 ~limlnated I . . ; : . , . . .
. . ; ui the pmce~lurc described in. . .
' ~ - Part rI: ', ~ , , ' , . :'~ y - .
~ '.: ' ', ' ' . : 1 ' ' ; . , .' _ ; ';- ' , :> . , , . . , _ . , ' 1. , , , . ' .
' ' . . ' ; KNA.yiald;ach,of tho~c parts, 2Ul28th~m ~ti~e efRNA
~-;15 is ratio ofttl~ 1~A, (an! -.

. ,. ~ ; '. ~~~,~~.a~-time PCIt assays based on'~Y8)~~C~reea, .~ ' sail' ,,far the ho t,~.ek, oeping gees . , ' ~ . ~ , ' :

- , . 'P~B~GD and 5' ~ priu~s are shown iu Tablc~s and ~res~ri#~ are':
' ' iwxth 2 beYow.' ~aoed~ . . ' . 3 -. ' ' ~: ~ ~ ~ ~ple ,'! , ~ ~ ' ' '.. ' ' : ~ " , ' . , ' ' ' ' . :clpsea'bet~. ; , .
' ' . , ~
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v .: . . . . , i'' : - . ' ~ - , , ~?I~Tp~'', k ~ y ~ . .
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:~~",~ , . . 'y 1 . . : , . , ' ' ' . , . ; ' ~ -, rv~:r.': : . ; . . , . !
i5~'': . , ' . . . . - , . , ~a <.1~.,'" i . . . , , I
, ' . , ' ?.y " i '.'~~r '5 ' . . . " .
_~ ~ . , . . . '; .

.'a ' . . . I ~ ' , .' . , i '':k.: , . . . v.r ' ,' . , .. , , ' , ' . , , . . ~ . . 'W ' ..

.. ' ' . , .~ .: , :i ~ , . . . , . s~ ' . , ''w'.'h '~ ' , ",~ '~'.', . .., ' ~ ' ~ ~ ~ . . ' ; ,~ _ I , ~ . ~ ~ , .~ , :'" .~ly'S r~.: 4. '. ~' 1 ~ ~ ' . ~ . . ' . . . ~~ ~ r~.: ~~: ~,''y''-.
~ 1 , . , . . - , . , ' .'~~l, ~- , :r" ';;~iL: ;~v ::.p ~ ' ~ . . . ~ . t . . . , ',-: ~'~'Y ', :.. i~ .. ,. ~ ' . . . . . . ~ , , ','' 4. ~ : ~ . . , , , '1 , , ., :_ ~: 4':;:_~_1 .:." r.p . : . . . . '. ~ , , .o ' : , .
~~~~,,"j,:, ~'~;, ~' a" ~ .' ;~o~l'setlVo: S-5005 . ' , , .. . ~ . , . ,' . ' . . , F,;, .' 1 I!5' '~..'.' , ~ .-'. ' ~.1~, n ~ .. ' ' . ' 1 ~W ' ' ~ ' , ,_. ; ~ ,~ . ,: i~ .. ~ . , ~'' 'Table ~. RN csxtraetion #kom $ bx'east a~ , . : ' i ~,:i~~r~"1;'~ '''~'.''. . . ' ~ , ~ ' ,. . . . - .
' ~.~ i..' it~'_'''~ . , ' . . ' ~ , . , . .. ' ' . , ' ' .~; ,". . . . ' : ' ..~'t~.~ '': ~.~d:~' ~' ~ ~ , ' . . ' . ~ , , - . I . .. -. ~ ' , . , .
~-' :;a' ~. , ',,, , ' '... ,GCLNC- Protvcot. Total RNA . 'RNA' ~ PBG~ ~'' PBC~D~~''' 'NIG,~t'~° , ' :~. ~.... ~ . .: , . . ~ . ~ . . , . , ~ DesCt'ip~on . Yield. . . ~ ..,G'~~ , Ct . . . .
~. ~~,pi~ . . ' ' ' ~vlNa) '. ~ . ,~~~~~ ~2soysa~ t ~ . , . : ;.': - ~ , .:., :~.. ~,. . - . . .
;;..~F:.:~ - .~;'~- ' , ,s, 'r ~. ' ' 1 ' ~ lClt M~nufBCtu~r's 3't.g2., 2.1 ..~27.~1r~3" . ~ .85' '~3,ZB' ~.' , ,, : _ '' ~::~ r :~...,. :_' :t"~:.. . . ; , -.i~~~ , ~ , , ~ . . ~ . . , ,, ~
. ' ~ '' . . , ' :, ~~... ° ' '"_' .'P . ' ~ aA Cent~fugatlon limm ; 90.~ , 2:1 . ' ' ~a7.5.1 ~ :as.4 . -' ~a~.a ;;; , ~ ~. '.~; f;, ~.r . _v ~' . . . , . . . . ~ ~ .
- ~ . . reduced' ~ I ' '~' , I' A' ' . ~ ~ ~ ' . . gg I~EGm~nat 1 RPE ' 28;i 1~ , , Z.1 ~ . X7.08 1 29.2T ~ ~ 28.09' ~ ~. .'' , '.:< ~ ~ s :; . . '' WeBh ' , , . .1 . . ' ' '';, ' . a ,°~:".'. .. .~ ; -, , . ' _ 2C ' Used vINR mini- 27.91 ' 2,1 ' 2~.1~ ,8,51 .. , ' 'v';,. ~.';° _v: ~ 1 . , ~ ' ' t~liCrdt4~9e~' ' v ' . . . : . ' '~ ' :w:;'' , .. , ,. . ' , ' ~ ' ; ~.; , ' ' 2.1 ' 27 3 49.$9 ~,3.Oi : . , ~ . _. 217 - ,~llminated'oolumn ' 95.1 . , . ' ~ .' . . , . .
. . . '~ ~., ,; , - , ' ' , drying step . . ' : . v . ~ .. _ , , ~ ''' I ' ' ' 2~1 ' -Modi9~fions 1-2 ~ 25.5 ~.1 27sC9 ~ . ~28,77. ~,'B~.~B '.;
, . ' ' ; ~,'.v, ~' ,,a .;.~ " . ~ , made (ells, ~ ~ . ~ . i . , ;'. 3. : = . . .
1 ,: agc'~ pos~trv ~ , ' wA. ralA ' , , ' nvA ' , 3G.1 ~ ' , 26.98 a~a. , ~'' : c~~:.i ~ ,~ . . . ',' '°. . . . ~ . . 1' . ~j.~ ;~' ~ , ~
:_I' ~. '.. . . _ ~' ~~,TCneg8tlV6 ,' , ' (~11A . NIA , . NIA_ .4B.0~.' ~ 60 .
, ~
"I ~ s', ~,' ,r ~~ ~ ' , ~ ',r ~~ ~ I : ; . 1 . , - ' . . j . ' ~ , '' . ,' v ;
.: i: :~ , ~. : ' . - , " ~ . ' ' ' i .. . . , ; .
..:.iv.~r S~ 1~ ~ ...:,~~;',y ,;~ .. ~.,'1~ , ~ , , ' - . ~ . ~ ' . ~ , , . .
. . ' 1, . . ' s ~ ' 1'.. ~.I~ . , .~ :' ' ' . . ~ .
~'. r. ,. . ~ ' , , ~
' ~Rsa~lts a~' cxp'e;dm~te iadica'ce tl~a't pro~col which, i'ur~aase ~s~eed ;~tnd lower ' , ' ~
~ ' --, ~ I 1.. : ~ ~ ' lns.eat ca is xrrsultad is aamo lQea~of yicld'aF'ItN~s~. However, the amn,mt~ol'~1'A v -a.',.~,;:' :' . , ~' -. .r ; ~. . .. . '' ' ~ ' ~, .. .. ' 1 _ ~iiald~d by ~acx~e~d ~sotoool is wall; aban!a that whicb'is reguirOd for ~
~ev~ ~ , , ~
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u..', All other t~e~,geats were ,~'roax .' oma~ae~ai . ' , , '' :
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of 25mM
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of Ia~daM
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from Each nadey:
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After caking with ' ' ~ a~
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le of , i~ .
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f _ t t S ~ ~ ~7('.
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e~'n:. . ' ;.:~t.f'~ ~~, .
'' omo~eaate' ~ ' ~'' ,~', ~
:. ' ~ trr.oa'ly 3tt eecpnds yield's .' acceptable RrTA, extraotioa.f.
; ' . , ~
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' .' . 5 _ . of homogEnate ,.
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t~: RL~' node was homogenized., Six l~dradmir.~oli ~ . ~~
~~ ~ tiu ' .
~' ~f ,',~' ' ~.
' .

'~ , . g modificdticrds asv' ~' , . ' ~': '. ..
.v~' w .~.' as'~samovoand RNA;was cxtraGted.uaia~ all 1 ~Lhe prOtocol:.slit~
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' . ' Is 2. A,s a cvatroi, another was irdatQd us~g , ~ ' abed i~r fi0~ ~Z of, l~tuag ~H_;
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~ ~ ' ' ~,.trifizgatiai NA tom each .. . ' sd._.. w~s.~sed iaatead o'F ~tha QIAshredder . eolua~a. : The ~
''.
' , .' ' . 10 ' .; ~rcg~oriarse,transoribed ~d c~usatifiied ' } wa$. as de$cxibed in ~x'az ~ple l..RealtimE"
, .
, , .~
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' ' _ pC&'vvas , ''..:'; per ..
~' , '~~.: ormad~using Taqtnaa grabes as ' descrt"bed ~~eviously,'i~ example . 3. . ' . , , t ' ~

:. . ~ .'~a ' , ' ' . .. ~ ' , ' , .

, , ' , s~ studies i~di~ted similar ltNA 'm the proG~col ' , ". of th yields of 0.31.Ng/l.yLj ~, ' , ' Results ' .
v ' ' .
j ~ ' : invtilwn~ng'tha~rAshredderr pmtoCol as ~comp~ed ~e centr~'ugationi ~ to 0.34 ~g/lt'L, in tl '~~ ~ . ~ ~- -, ' I

~~ ~~ .. ~h '~ 2~28Qr~n ratio's, I5f 2.1 v~ere~mglcs.
iadiaatin~g.
~' obtanned'ia both s '- ' ' '= .
~ ~
~' s~
.
' '::
~
', ' . . . walues.far ~:':..' ' ' ~ liigh ~ A.-' Rea1:time PGR assays also ..''~' guality. indicated'si~ail~r Ct "~

- , ~ ~ . . ~ . .. . . ~ . .. . .
;~_a . :~ . . ' wi'th RNA extracted by the. ,ompa~d to~t~ . . ;
' ' Max~naglo'6'~IAsbn~dder'aohmma ag:'~
.

.

. , '~ pi'otoc.~ilvii (22.5 versus 22;0), a8 wall ' ,'.'': invo ag'vvith.B305A (2~r.7 bus 26.6'),as well:
~' ~'' , ' .: ; . ~;' ~ ' keeiiiag genes FBGT3 (29.1 versa~:29.0);~ta (18.5 versus ' '.:' y~t'h the~h and. 8-ac , , - . . ;.
, .
.
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, , , ' I
' .; . ' ; quality . . -''' gyp' summ exper~ia~eatiadicatea s~m~arRNA d real-time PCR, ' ' yield;
. ~s ' ' . . rotocol i~avoivi~tg'a QTAsbrei~er~subs~tituted for a 3min ~-';' ' ~ is ~rith the .
,:,':. n '' ti ,:,'~
:
;

' p .
ca .
o ti ' et~rp' af~c, homogeai~atioa. , 'This modi~rcala4>a reduces ' . ! the time reqdited to . ~25 ~. ~ is important ire deve _ ~ perform RN extr'dCtioa'h~ ~.S minutes, avl~ieh~Opirig P~'t~Q018'' ~ h ' ' ' ' .
i', v .r , : " _ ' rable~for ~ 'geratiie ex~ othar applitatiCrnBl~.are requited tu:
gv9 in' WhiCii ia'st ;' . , .
~w ~ ~: ' cane. ~ ' , '' ' ~ ~ ' ~.
.' . . ' , pact ' . . ' , ' , ' ~~~.f pa~i . ' w.~ . .~.5~..J. ' . . . . . . ' . . . ' . .
.

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n iy i . -. r1 '' ~ .' , i s. . - ~ ExaxZlple -~ 5. ..
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' rrmparison ~f Rapid RNA
e~tractioa method axed ste~lsrid (Prior .Ait~
~' :
.
;
.

';
':
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.'~ w; ~. ~ ~t~~it~EN, : . ~a pro v~

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col an Colon Tissue ' , ' .'..."~ ~
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v ~

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. _ ~o~lo , ' : ~ ' ~ .
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exat~p~e caxapares the fan cf RNA
fi'~m ooloti't~i~~ae with'biotli'~e~

.
' :
:

- ~
r :, "
.
:
.
.

, .
:method .
of th iuv~ition sad fihe prior arC.
Tw~.cy mg, o~
co'Lr~a tiseuywi~
added to .

' , ' d~00u1 ~.RLT
of H aadhomogenized as descri'bediu l3xamvpla.3.
~tlC-s~mple ;0f ' ~
, ' -, .

_ .
. i~oraogenizae . , ~calai~'ca"cer tissue~w~s tr~.ted acce~rdin the ~1' ~
_ ~' ~
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~ ~ ~e$Qribed, ' :' ~ is ;~ . ,' ~ ,.
: 10 ~
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xamlsle 3,A, ~d Que sa~aople wad treated acciu'ding toa~e.~IA.C'-.~
~
#~1' _ ~ , ~protoso~.
i' a 1 . '. as, ~ , ~ .' . . .
. - crib~d is Example;3, part 8.
Re&ultant R1~FA
fra~a'bo;6a ~
wss ',(':i: ~ . , . . w- ~ .
. . , : S , 011 t)10'w i ' - . ' . u;t~:. . ' .
' ' . .
.
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$io8aalyZi.r.
.

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' ~le~trcrpl~era-v ' ' - s~~or RNA
~attv~rith the rapid ~ttLad'a~.~
tl~
isrioor ark ehow~ed ' ~
.
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.

' ''that ,..
~' ~ ' t~ R~NN~r .
' . 1~. , .
o b~
aempl~
processedwith tile ~IA~"rE~T
piatocol'~~das 1.~4' sad the '..-.
~
~
''"1.

:' . ; ' . . ,ple 'w1'th . t~.
rapid'protocei was 1.3f.
Generally;
any sa~plewvith a~
~1'A;-''F
:
' ;

,. , ; .
a.' ; ' ~tio ~a~~ , ~' ;1' -~ r . ~s . . . ; .~a~a~a , . . . ~ep~io.
Ag a~c~a by ~~
~~~aioa~l:rh~.
' ...

. ' ~ . .
.
.
' '''~ ' , . ~.~atl~ple .
: prose , ~ - sad vyith. rapid protocol had as ItNA yield of 136ug1m~ 'ocher-eas~ttve . ' , ,'y~pl8 , .. traa , r. ' . .
Wlth the (~~AA~T
method had a yield Of Z~Oag/mt.' 1'~'he yield ~Oht$me~
' .
, , .,; . . - .
. :: ' ~ ~;y. ol-is well a'bava that which . . ~ . '..;;both. is required far~a rdverse. '.ptase ' 1 ::; , pinto z~aotiou, ~ . . . . .
~0 _ ,' , : . - ~ S
' ' , ' ~ tr0 ~.~u~~1 : . . ' ' . , ~ ' .
' ' ..
' .
.
' , , ' ' 1, k" ~;' li~ , . ~ple .
' - ~ 1' ~ 6, ' , ' ' ; ~
. ' .
' ' I
' ' ',' ..
-~
-~.
iy ffeot ~of' Ihititin~
ttf Lysste c~a RNA
yield and pi~cisi~
, . T'hc fallow,, ' .
~f ,_ ~ ~ ~' ~ , , , ' . , , example illustrates the effect of dilu#ox~
~'~
lyaate ~aa R'1~?A
yield.
and :.
'' ' - - i', ~~ . ~ '-pr~cisiotzA
, - :; ;~ of . recovery.
, rn ' 25' this ,example;
20a~g, lUrag'or ,mg.a~
~ozeu pig node' ' ~
', .
' .
.

';' ~ . ' ~ieeuo Tissue ~ ' , - . vcras was cu ground using a SOmt ~isgosab~
Tissue ~
' ' for sac, ' ' .

' ' a:' y W d, fih~e - .
' , : - . ~amp1' ~ were yattexed; 3'hree t~ep?,ic,~tes ~ ~rere per.Eormeri fear each.aoda~w~gjit,, ~ v.~
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., ; , -_' : _ ' ' v : , . , ~ .. ' as then 1' T . ' ' ' '- , . ' ' '~ ' ~ v hich .I . ~
~ .
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~f'' t ' c~
, i' ' u l ~ ;r ~ zh.1 of a :
. a o w ' one uge w ' u ' a ' h'lyeatc r~ra~
~d to a 1.7 m : , ~ . , ' ~
r :;~,:' .
' a:' ~" ae~ai~iftigeda~ '1~'fos 30 sec.
~ ;, ' 8ppeudnrFmiCmftige ' ~ at ' ~ . .
;., ~ maximum - sped (Z~tO~UI~

~' ~ , as 'w , oen'tdfaged , . 1 'v~ thraug~e i ' h a ', , < Q~~e'~ olvamn sad~e-~~, liters ' ~

:j , ven, w , , :--.:.'~~: F. , ~ed at maximum speed for 30 ago. .
; ;.. -s 'fhe column ' ies were cend:'iflt vvsa tl~, washed, ~
~w"' ' ~ : ~

, p , , ..
~ ; '' 1 ~
~

, . . " : , ., ; ' 'tJa~'colu~z'~d, Ie 2, plait I
steps '~. and 4.- :
:~' 3,tNA ' - : ': ' , S eluted ~;.~.. as deecrlbed'provioualy iz~.:Bx ~
l . r;', ~ y; yA ~e re .slud'ces a~
' ' ,. was ~ z' ,', . by ' .' ..' meaa~a : . of ' a . C'e~ie Spec lz~
' R.aeults of : t , , . ._. , , ,. . . , G:;' : , .
;.: . ia T eci~of~ at au 'initial ao p ~ ,;;,;:'... b atleut t~ague-waight of ' ' ~ d illusfr ~..~ ex . t . ~
, ' ~ a pi , .
~bwn. v ith a ~
e~ e a , sa ~
e ' . Z.SmS r, as :camp~r d ,ta ssm homo Ou~l of 3ta~geni~atioa buff l ~u~ad is~. a w .~ ' ~' -~~~et, ' wei ~ .
~ 'these ~' re.~ults ;:~.r~; indir~te v.: that.
: .
~"' , w ' , ' ;
y ' , inaludit~~
lOmg axtd ~~mg uotle ~tiss~ie, , . , :. .

, '~ . - Bion ' . 'i~op~ed off ' , ' ' p RNA , . necav~ery. weight, Also, n ;
. can b~
obtained at lower noc~

, -, would. ted that more dilute homogeliatcs, ~ be e~cp would be~~.ltei~ca:~ -''1'r~ ' ~ , , ~ ster through the , .
, . ' . . . '~fil~r'clog~iu~,' ', . ' .;.onTumn,, _ ' ' v , .r., aad; there - would be ~~ oven .. fevYerpotential p~obla~s wit~.
:

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: . ' 1.'i. , , , ' ' . ~ , , ~ ' ' : ~ k ~ ' B~' 1 . ~ .' : ~ 'Table oa of'Lysate pxt RNA recovery ' ~ .
5 , ~ t . , 'E of l~luti . .

. .. - . , ' , , -. . "

~ .; ' , Tvtal FtNA i~ec~v~ry:' R~pf~ Ea~r~~lQnllniqi.~~1 ' ' : '~e~a .. ' .' , 1 ' :.
.. r , ; H
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, . ' ., ' , .. ,. .' .. , CA 02463716 2004-04-30 ' SEQUENCE,LISTING
<110> Veridex, LLC
<120> RAPID EXTRACTION OF RNA FROM CELLS AND TISSUES
<130> 1011-4415CA
<140> 10/427,217 <141> 2003-05-O1 <160> 35 ' <170> PatentIn version 3.1 r210> 1 <211> 27 <212> DNA
<213> Artificial <220> ' <223> Artificial Sequence is a Primer <400> 1 tcccatcaga atccaaacaa gaggaag , 27 <210> 2 <211> 26 <212> DNA ' , <213> Artificial <220>
<223> artifiical sequence is a primer <400> 2 ggctgttgct tggacttctc taaaga 26 <210> 3 <211> 29 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a primer <400> 3 caaacggatg aaactctgag caatgttga 29 <210> 4 <211> 24 <212> DNA
<213> Artificial <220>

<223> Artficial sequence is a primer <400> 4 tctgtgagcc aaaggtcttg caga 24 <210> 5 <211> 23 <212> DNA

<213> Artificial ' ' <220>

<223> Artificial sequence is a primer <400> 5 ggccaacaaa gctcaggaca aca 23 ' <210> 6 <211> 25 ' <212> DNA

<213> Artificial <220>

<223> Artificial sequence is a Primer <400> 6 gcagtgactt cgtcatttgg acgta 25 <210> 7 <211> 36 <212> DNA

<213> Artificial , ' <220>

<223> Artificial sequence is a Probe <900> 7 ' tgtttatgca attaatatat gacagcagtc 36 tttgtg <210> 8 <211> 20 <212> DNA

<213> Artificial <220>

<223> Artificial sequence is a Primer <400> 8 ctgaggcacc tggaaggagg 20 <210> 9 <211> 20 <212> DNA

<213> Artificial <220>
<223> Artificial sequence is a Primer <400> 9 catcttcatg ctgggcaggg 20 ' <210> 10 ~ ' <211> 29 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Primer ' <400> 10 ctgaggcacc tggaaggagg ctgcagtgt 29 <210> 11 ~ ' <211> 21 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Primer <400> 11 tctgataaag gccgtacaat g 21 <210> 12 , ' <211> 22 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Primer <900> 12 tcacgacttg ct~tttttgc tc 22 <210> 13 <211> 26 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Probe <400> 13 atcaaaaaac aagcatggcc tacacc 26 <210>

<211>

<212>
DNA

<213>
Human <400>

atggtggttgaggttgattccatgccggctgcctcttctgtgaagaagccatttggtctc60 aggagcaagatgggcaagtggtgctgccgttgcttcccctgctgcagggagagcggcaag120 agcaacgtgggcacttctggagaccacgacgactctgctatgaagacactcaggagcaag180 atgggcaagtggtgccgccactgcttcccctgctgcagggggagtggcaagagcaacgtg~240 ggcgcttctggagaccacgacgactctgctatgaagacactcaggaacaagatgggcaag300 tggtgctgccactgcttcccctgctgcagggggagcagcaagagcaaggtgggcgcttgg360 ggagactacgatgacagtgccttcatggagcccaggtaccacgtccgtggagaagatctg420 , gacaagctccacagagctgcctggtggggtaaagtccccagaaaggatctcatcgtcatg48'0 ctcagggacactgacgtgaacaagcaggacaagcaaaagaggactgctctacatctggcc540 tctgccaatgggaattcagaagtagtaaaactcctgctggacagacgatgtcaacttaat600 gtccttgacaacaaaaagaggacagctctgataaaggccgtacaatgccaggaagatgaa660 tgtgcgttaatgttgctggaacatggcactgatccaaatattccagatgagtatggaaat720 accactctgcactacgctatctataatgaagataaattaatggccaaagcactgctctta780 ' tatggtgctgatatcgaatcaaaaaacaagcatggcctcacaccactgttacttggtgta840 catgagcaaaaacagcaagtcgtgaaatttttaattaagaaaaaagcgaatttaaatgca900 ctggatagatatggaaggactgctctcatacttgctgtatgttgtggatcagcaagtata960 gtcagccttctacttgagcaaaatattgatgtatcttctcaagatctatctggacagacg1020 gccagagagtatgctgtttctagtcatcatcatgtaatttgccagttactttctgactac1080 aaagaaaaacagatgctaaaaatctcttctgaaaacagcaatccagaaaatgtctcaaga1140 accagaaataaataa 1155 <210> 15 <211> 3865 <212> DNA
<213> Human <400> 15 tccgagctga ttacagacac caaggaagat gctgtaaaga gtcagcagcc acagccctgg 60 ctagctggcc ctgtgggcat ttattagtaa agttttaatg acaaaagctt tgagtcaaca 120 cacccgtggg taattaacct ggtcatcccc accctggaga gccatcctgc ccatgggtga 180 tcaaagaagg aacatctgca ggaacacctg atgaggctgc acccttggcg gaaagaacac 240 ctgacacagctgaaagcttggtggaaaaaacacctgatgaggctgcacccttggtggaaa 300 gaacacctgacacggctgaaagcttggtggaaaaaacacctgatgaggctgcatccttgg 360 tggagggaacatctgacaaaattcaatgtttggagaaagcgacatctggaaagttcgaac 420 agtcagcagaagaaacacctagggaaattacgagtcctgcaaaagaaacatctgagaaat 480 ttacgtggccagcaaaaggaagacctaggaagatcgcatgggagaaaaaagaagacacac 540 ctagggaaattatgagtcccgcaaaagaaacatctgagaaatttacgtgggcagcaaaag 600 gaagacctaggaagatcgcatgggagaaaaaagaaacacctgtaaagactggatgcgtgg 660 caagagtaac atctaataaaactaaagttttggaaaaaggaagatctaagatgattgcat720 gtcctacaaa agaatcatctacaaaagcaagtgccaatgatcaga~gttcccatcagaat780 ccaaacaaga ggaagatgaagaatattcttgtgattctcggagtctctttgagagttctg840 ' caaagattca agtgtgtatacctgagtctatatatcaaaaagtaatggagataaatagag900 aagtagaaga gcctcctaagaagccatctgccttcaagcctgccattgaaatgcaaaact960 ctgttccaaa taaagcctttgaattgaagaatgaacaaacattgagagcagatccgatgt1020 tcccaccaga atccaaacaaaaggactatgaagaaaattcttgggattctgagagtctct1080 gtgagactgtttcacagaaggatgtgtgtttacccaaggctacacatcaaaaagaaatag1140 ataaaataaatggaaaattagaagagtctcctaataaagatggtcttctgaaggctacct1200 gcggaatgaaagtttctattccaactaaagccttagaattgaaggacatgcaaactttca1260 aagcagagcctccggggaagccatctgccttcgagcctgccactgaaatgcaaaagtctg1320 tcccaaataaagccttggaattgaaaaatgaacaaacattgagagcagatgagatactcc1380 catcagaatccaaacaaaaggactatgaagaaagttcttgggattctgagagtctctgtg1440 agactgtttcacagaaggatgtgtgtttacccaaggctrcrcatcaaaaagaaatagata1500 aaataaatggaaaattagaagggtctcctgttaaagatggtcttctgaaggctaactgcg1560 gaatgaaagtttctattccaactaaagccttagaattgatggacatgcaaactttcaaag1620 cagagcctcccgagaagccatctgccttcgagcctgccattgaaatgcaaaagtctgttc1680 caaataaagccttggaattgaagaatgaacaaacattgagagcagatgagatactcccat1740 cagaatccaaacaaaaggactatgaagaaagttcttgggattctgagagtctctgtgaga1800 ctgtttcacagaaggatgtgtgtttacccaaggctrcrcatcaaaaagaaatagataaaa1860 taaatggaaa attagaagag tctcctgata atgatggttt tctgaaggct ccctgcagaa 1920 tgaaagtttc tattccaact aaagccttag'aattgatgga catgcaaact ttcaaagcag 1980 agcctcccga gaagccatct gccttcgagc ctgccattga aatgcaaaag tctgttccaa 2040 ataaagcctt ggaattgaag aatgaacaaa cattgagagc agatcagatg ttcccttcag 2100 aatcaaaaca aaagaasgtt gaagaaaatt cttgggattc tgagagtctc cgtgagactg 2160 tttcacagaa ggatgtgtgt gtacccaagg ctacacatca aaaagaaatg gataaaataa, 2220 gtggaaaatt agaagattca actagcctat caaaaatctt ggatacagtt cattcttgtg 2280 aaagagcaag ggaacttcaa aaagatcact gtgaacaacg tacaggaaaa atggaacaaa 2340 tgaaaaagaa gttttgtgta ctgaaaaaga aactgtcaga agcaaaagaa ataaaatcac 2400 agttagagaa ccaaaaagtt aaatgggaac aagagctctg cagtgtgaga ttgactttaa 2460 accaagaaga agagaagaga agaaatgccg atatattaaa tgaaaaaatt agggaagaat 2520 taggaagaat cgaagagcag cataggaaag agttagaagt gaaacaacaa cttgaacagg 2580 ctctcagaat acaagatata gaattgaaga gtgtagaaag taatttgaat caggtttctc 2640 , acactcatga aaatgaaaat tatctcttac atgaaaattg catgttgaaa aaggaaattg 2700 ccatgctaaa actggaaata gccacactga aacaccaata ccaggaaaa~g gaaaataaat 2760 actttgagga cattaagatt ttaaaagaaa agaatgctga acttcagatg accctaaaac 2820 tgaaagagga atcattaact aaaagggcat ctcaatatag tgggcagctt aaagttctga 2880 tagctgagaa cacaatgctc acttctaaat tgaaggaaaa acaagacaaa gaaatactag 2940 aggcagaaat tgaatcacac catcctagac tggcttctgc tgtacaagac catgatcaaa 3000 ttgtgacatc aagaaaaagt caagaacctg ctttccacat tgcaggagat gcttgtttgc 3060 aaagaaaaat gaatgttgat gtgagtagta cgatatataa caatgaggtg ctccatcaac 3120 cactttctga agctcaaagg aaatccaaaa gcctaaaaat taatctcaat tatgcmggag 3180 atgctctaag agaaaataca ttggtttcag aacatgcaca aagagaccaa cgtgaaacac 3240 agtgtcaaat gaaggaagct gaacacatgt atcaaaacga acaagataat gtgaacaaac 3300 acactgaaca gcaggagtct ctagatcaga aattatttca actacaaagc aaaaatatgt 3360 ggcttcaaca gcaattagtt catgcacata agaaagctga caacaaaagc aagataacaa 3420 ggcttcaaca gcaattagtt catgcacata agaaagctga caacaaaagc aagataacaa 3420 ttgatattca ttttcttgag aggaaaatgc aacatcatct cctaaaagag aaaaatgagg 3480 agatatttaa ttacaataac catttaaaaa accgtatata tcaatatgaa aaagagaaag 3540 cagaaacagaaaactcatgagagacaagcagtaagaaacttcttttggagaaacaacaga3600 ccagatctttactcacaactcatgctaggaggccagtcctagcatcaccttatgttgaaa3660 atcttaccaatagtctgtgtcaacagaatacttattttagaagaaaaattcatgatttct3720 tcctgaagcctacagacataaaataacagtgtgaagaattacttgttcacgaattgcata3780 aagctgca~caggattcccatctaccctgatgatgcagcagacatcattcaatccaaccag3840 aatctcgctctgtcactcaggctgg 3865 <210> 16 <211> 3282 <212> DNA
<213> Human <400> 16 gggacagggc tgaggatgag gagaaccctg gggacccaga~agaccgt,gcc ttgcccggaa 60 gtcctgcctg taggcctgaa ggacttgccc taacagagcc tcaaQaacta cctggtgatt 120 ' cctacttcag ccccttggtg tgagcagctt ctcaacatga actacagcct ccacttggcc 180 ttcgtgtgtc tgagtctctt cactgagagg atgtgcatcc aggggagtca gttcaacgtc 240 gaggtcggca gaagtgacaa gctttccctg cctggctttg agaacctcac agcaggatat 300 aacaaatttc tcaggcccaa ttttggtgga gaacccgtac agatagcgct gactctggac 360 attgcaagtatctctagcatttcagagagtaacatggactacacagccaccatatacctc420 cgacagcgctggatggaccagcggctggtgtttgaaggcaacaagagcttcactctggat480 gcccgcctcgtggagttcctctgggtgccagatacttacattgtggagtccaagaagtcc540 ttcctccatgaagtcactgtgggaaacaggctcatccgcctcttctccaatggcacggtc600 ctgtatgccctcagaatcacgacaactgttgcatgtaacatggatctgtctaaatacccc660 atggacacacagacatgcaagttgcagctggaaagctggggctatgatggaaatgatgtg720 gagttcacctggctgagagggaacgactctgtgcgtggactggaacacctgcggcttgct780 cagtacaccatagagcggtatttcaccttagtcaccagatcgcagcaggagacaggaaat840 tacactagattggtcttacagtttgagcttcggaggaatgttctgtatttcattttggaa900 acctacgttccttccactttcctggtggtgttgtcctgggtttcattttggatctctctc960 gattcagtccctgcaagaacctgcattggagtgacgaccgtgttatcaatgaccacactg1020 atgatcgggtcccgcacttctcttcccaacaccaactgcttcatcaaggccatcgatgtg1080 tacctggggatctgctttagctttgtgtttggggccttgctagaatatgcagttgctcac1140 tacagttcct tacagcagat ggcagccaaa gataggggga caacaaagga agtagaagaa 1200 gtcagtatta ctaatatcat caacagctcc atctccagct ttaaacggaa gatcagcttt 1260 gccagcattg aaatttccagcgacaacgttgactacagtgacttgacaatgaaaaccagc1320 gacaagttca agtttgtcttccgagaaaagatgggcaggattgttgattatttcacaatt1380 caaaacccca gtaatgttgatcactattccaaactactgtttcctttgatttttatgcta1440 gccaatgtat tttactgggcatactacatgtatttttgagtcaatgttaaatttcttgca1500 tgccataggt cttcaacaggacaagataatgatgtaaatggtattttaggccaagtgtgc1560 acccacatcc aatggtgctacaagtgactgaaataatatttgagtctttctgctcaaaga1620 atgaagctcc aaccattgttctaagctgtgtagaagtcctagcattataggatcttgtaa1680 tagaaacatc agtccattcctctttcatcttaatcaaggacattcccatggagcccaaga1740 ttacaaatgt actcagggctgtttattcggtggctccctggtttgCatttacctcatata1800 aagaatggga aggagaccattgggtaaccctcaagtgtcagaagttgtttctaaagtaac1860 ' tatacatgtt ttttactaaatctctgcagtgcttataaaatacattgttgcctatttagg1920 gagtaacatt ttctagtttttgtttctggttaaaatgaaatatgggcttatgtcaattca1980 ttggaagtca atgcactaactcaataccaagatgagtttttaaataatgaatattattta2040 ataccacaac agaattatcc ccaatttcca ataagtccta tcattgaaaa ttcaaatata 2100 agtgaagaaaaaattagtagatcaacaatctaaacaaatccctcggttctaagatacaat2160 ggattccccatactggaaggactctgaggctttattcccccactatgcatatcttatcat2220 tttattattatacacacatccatcctaaactatactaaagcccttttcccatgcatggat2280 ggaaatggaagatttttttgtaacttgttctagaagtcttaatatgggctgttgccatga2340 aggcttgcagaattgagtccattttctagctgcctttattcacatagtgatggggtacta2400 aaagtactgggttgactcagagagtcgctgtcattctgtcattgctgctactctaacact2460 gagcaacactctcccagtggcagatcccctgtatcattccaagaggagcattcatccctt2520 tgctctaatgatcaggaatgatgcttattagaaaacaaactgcttgacccaggaacaagt2580 ggcttagcttaagtaaacttggctttgctcagatccctgatccttccagctggtctgctc2640 tgagtggcttatcccgcatgagcaggagcgtgctggccctgagtactgaactttctgagt2700 aacaatgagacacgttacagaacctatgttcaggttgcgggtgagctgccctctccaaat2760 ccagccagagatgcacattcctcggccagtctcagccaacagtaccaaaagtgatttttg2820 agtgtgccagggtaaaggcttccagttcagcctcagttattttagacaatctcgccatct2880 ttaatttcttagcttcctgttctaataaatgcacggctttacctttcctgtcagaaataa2940 accaaggctctaaaagatgatttcccttctgtaactccctagagccacaggttctcattc3000 cttttcccattatacttctcacaattcagtttctatgagtttgatcacctgattttttta3060 acaaaatatttctaacgggaatgggtgggagtgctggtgaaaagagatgaaatgtggttg3120 tatgagccaatcatatttgtgattttttaaaaaaagtttaaaaggaaatatctgttctga3180 aaccccacttaagcattgtttttatataaaaacaatgataaagatgtgaactgtgaaata3240 , aatataccatattagctacccaccaaaaaaaaaaaaaaaaas 3282 <210>

<211>

<212>
DNA

<213>
Human <400>

gacagcggcttccttgatccttgccacccgcgactgaacaccgacagcagcagcctcacc 60 atgaagttgctgatggtcctcatgctggcggccctctcccagcactgctacgcaggctct 120 ggctgccccttattggagaatgtgatttccaagacaatcaatccacaagtgtctaagact 180 gaatacaaagaacttcttcaagagttcatagacgacaatgccactacaaatgccatagat 240 gaattgaaggaatgttttcttaaccaaacggatgaaactctgagcaatgttgaggtgttt 300 atgcaattaatatatgacagcagtctttgtgatttattttaactttctgcaagacctttg 360 gctcacagaactgcagggtatggtgagaaaccaactacggattgctgcaaaccacacctt 420 ctctttcttatgtctttttactacaaactacaagacaattgttgaaacctgctatacatg 480 tttattttaataaattgatggca 503 <210>

<211>

<212>
DNA

<213>
Human <400> 18 gacagcggct tccttgatcc ttgccacccg cgactgaaca ccgacagcag cagcctcacc 60 atgaagttgc tgatggtcct catgctggcg gccctctccc agcactgcta cgcaggctct 120 ggctgcccct tattggagaa tgtgatttcc aagacaatca atccacaagt gtctaagact 180 gaatacaaag aacttcttca agagttcata gacgacaatg ccactacaaa tgccatagat 240 gaattgaagg aatgttttct taaccaaacg gatgaaactc tgagcaatgt tgaggtgttt 300 atgcaattaa tatatgacag cagtctttgt gatttatttt aactttctgc aagacctttg 360 gctcacagaa ctgcagggta tggtgagaaa ccaactacgg attgct,gcaa accacacctt 420 ctctttctta tgtcttttta ctacaaacta caagacaatt gttgaaacct gctatacatg 480 tttattttaa taaattgatg gca 503 <210> 19 <211> 1793 <212> DNA
<213> Human <400> 19 cgcgtccgcc ccgcgagcac agagcctcgc ctttgccgat ccgccgcccg tccacacccg 60 ccgccagctc accatggatg atgatatcgc cgcgctcgtc gtcgacaacg gctccggcat 120 gtgcaaggcc ggcttcgcgg gcgacgatgc cccccgggcc gtcttcgcct ccatcgtggg 180 gcgccccagg caccagggcg tgatggtggg catgggtcag aaggattcct atgtgggcga 240 ' cgaggcccag agcaagagag gcatcctcac cctgaagtac cccatcgagc acggcatcgt 300 caccaactgg gacgacatgg agaaaatctg gcaccacacc ttctacaatg agctgcgtgt 360 ggctcccgag gagcaccccg tgctgctgac cgaggccccc ctgaacccca aggccaaccg 420 cgagaagatg acccagatca tgtttgagac cttcaacacc ccagccatgt acgttgctat 480 ccaggctgtg ctatccctgt acgcctctgg ccgtaccact ggcatcgtga tggactccgg 540 tgacggggtc acccacactg tgcccatcta cgaggggtat gccc~tccccc atgccatcct 600 gcgtctggac ctggctggcc gggacctgac tgactacctc atgaagatcc tcaccgagcg 660 cggctacagc ttcaccacca cggccgagcg ggaaatcgtg cgtgacatta aggagaagct 720 gtgctacgtc gccctggact tcgagcaaga gatggccacg gctgcttcca gctcctccct 780 ggagaagagctacgagctgcctgacggccaggtcatcaccattggcaatgagcggttccg 840 ctgccctgaggcactcttccagccttccttcctgggcatggagtcctgtggcatccacga 900 aactaccttcaactccatcatgaagtgtgacgtggacatccgcaaagacctgtacgccaa 960 cacagtgctgtctggcggcaccaccatgtaccctggcattgccgacaggatgcagaagga 1020 gatcactgccctggcacccagcacaatgaagatcaagatcattgctcctcctgagcgcaa 1080 gtactccgtgtggatcggcggctccatcctggcctcgctgtccaccttccagcagatgtg 1140 gatcagcaag caggagtatg acgagtccgg cccctccatc gtccaccgca aatgcttcta 1200 ggcggactat gacttagttg cgttacaccc tttcttgaca aaacctaact tgcgcagaaa 1260 acaagatgagattggcatggctttatttgttttttttgttttgttttggttttttttttt1320 tttttggcttgactcaggatttaaaaactggaacggtgaaggtgacagcagtcggttgga1380 gcgagcatcccccaaagttcacaatgtggccgaggactttgattgcacattgttgttttt1440 ttaatagtcattccaaatatgagatgcattgttacaggaagtcccttgccatcctaaaag1500 ccaccccacttctctctaaggagaatggcccagtcctctcccaagtccacacaggggagg1560 tgatagcattgctttcgtgtaaattatgtaatgcaaaatttttttaatcttcgccttaat1620 acttttttattttgttttattttgaatgatgagccttcgtgcccccccttcccccttttt,1680 gtcccccaac ttgagatgta tgaaggcttt tggtctccct gggagtgggt ggaggcagcc 1740 agggcttacc tgtacactga cttgagacca gttgaataaa agtgcacacc tta 1793 <210> 20 <211> 1377 <212> DNA
<213> Human <400> 20 cacacagcct actttccaag cggagccatg tctggtaacg gcaatgcggc tgcaacggcg 60 gaagaaaaca gcccaaagat gagagtgatt cgcgtgggta cccgcaagag ccagcttgct 120 cgcatacaga cggacagtgt ggtggcaaca ttgaaagcct cgtaccctg~g cctgcagttt 180 gaaatcattg ctatgtccac cacaggggac aagattcttg atactgcact ctctaagatt 240 ggagagaaaa gcctgtttac caaggagctt gaacatgccc tggagaagaa tgaagtggac 300 ctggttgttc actccttgaa ggacctgccc actgtgcttc ctcctggctt caccatcgga 360 gccatctgca agcgggaaaa ccctcatgat gctgttgtct ttcacccaaa atttgttggg 420 aagaccctagaaaccctgccagagaagagtgtggtgggaaccagctccctgcgaagagca480 gcccagctgcagagaaagttcccgcatctggagttcaggagtattcggggaaacctcaac540 acccggcttcggaagctggacgagcagcaggagttcagtgccatcatcctagcaacagct600 ggcctgcagcgcatgggctggcacaaccgggtggggcagatcctgcaccctgagaaatgc660 atgtatgctgtgggccagggggccttgggcgtggaagtgcgagccaaggaccaggacatc720 ttggatctggtgggtgtgctgcacgatcccgagactctgcttcgctgcatcgctgaaagg780 gccttcctgaggcacctggaaggaggctgcagtgtgccagtagccgtgcatacagctatg840 aaggatgggcaactgtacctgactggaggagtctggagtctagacggctcagatagcata900 caagagaccatgcaggctaccatccatgtccctgcccagcatgaagatggccctgaggat960 gacccacagt tggtaggcat cactgctcgt aacattccac gagggcccca gttggctgcc 1020 cagaacttgg gcatcagcct ggccaacttg ttgctgagca aaggagccaa aaacatcctg 1080 gatgttgcac ggcagcttaa cgatgcccat taactggttt gtggggcaca gatgcctggg 1140 ttgctgctgt ccagtgccta catcccgggc ctcagtgccc cattctcact gctatctggg 1200 gagtgattac cccgggagac tgaactgcag ggttcaagcc ttccagggat ttgcctcacc 1260 ttggggcctt gatgactgcc ttgcctcctc agtatgtggg ggcttcatct ctttagagaa 1320 gtccaagcaa cagcctttga atgtaaccaa tcctactaat aaaccagttc tgaaggt ~ 1377 <210> 21 <211> 1524 <212> DNA
<213> Human , <400> 21 , ' agcagacaga ggactctcat taaggaaggt gtcctgtgcc ctgaccctac aagatgccaa 60 gagaagatgc tcacttcatc tatggttacc ccaagaaggg gcacggccac tcttacacca 120 cggctgaagaggccgctgggatcggcatcctgacagtgatcctgggagtcttactgctca 180 , tcggctgttggtattgtagaagacgaaatggatacagagccttgatggataaaagtcttc 240 atgttggcactcaatgtgccttaacaagaagatgcccacaagaagggtttgatcatcggg 300 acagcaaagtgtctcttcaagagaaaaactgtgaacctgtggttcccaatgctccacctg 360 cttatgagaaactctctgcagaacagtcaccaccaccttattcaccttaagagccagcga 420 gacacctgagacatgctgaaattatttctctcacacttttgcttgaatttaatacagaca 480 tctaatgttctcctttggaatggtgtaggaaaaatgcaagccatctctaataataagtca 540 gtgttaaaattttagtaggtccgctagcagtactaatcatgtgaggaaatgatgagaaat 600 attaaattgggaaaactccatcaataaatgttgcaatgcatgatactatctgtgccagag 660 gtaatgttagtaaatccatggtgttattttctgagagacagaattcaagtgggtattctg 720 gggccatccaatttctctttacttgaaatttggctaataacaaactagtcaggttttcga 780 accttgaccgacatgaactgtacacagaattgttccagtactatggagtgctcacaaagg 840 atacttttacaggttaagacaaagggttgactggcctatttatctgatcaagaacatgtc 900 agcaatgtctctttgtgctctaaaattctattatactacaataatatattgtaaagatcc 960 tatagctctttttttttgagatggagtttcgcttttgttgcccaggctggagtgcaatgg 1020 cgcgatcttggctcaccataacctccgcctcccaggttcaagcaattctcctgccttagc 1080 ctcctgagta gctgggattacaggcgtgcgccactatgcctgactaattttgtagtttta1140 gtagagacgg ggtttctccatgttggtcaggctggtctcaaactcctgacctcaggtgat1200 ctgcccgcct cagcctcccaaagtgctggaattacaggcgtgagccaccacgcctggctg1260 gatcctatat cttaggtaagacatataacgcagtctaattacatttcacttcaaggctca1320 atgctattct aactaatgacaagtattttctactaaaccagaaattggtagaaggattta1380 aataagtaaa agctactatgtactgccttagtgctgatgcctgtgtactgccttaaatgt1440 acctatggca atttagctctcttgggttcccaaatccctctcacaagaatgtgcagaaga1500 aatcataaag gatcagagattctg 1524 <210> 22 <211> 2384 <212> DNA

<213> Huma n <400> 22 tattgagttc ttcaaacatt gtagcctctt tatggtctct gagaaataac taccttaaac 60 ccataatctt taatacttcc taaactttct taataagaga agctctattc ctgacactac 120 ctctcatttgcaaggtcaaatcatcattagttttgtagtctattaactgggtttgcttag 180 gtcaggcattattattactaaccttattgttaatattctaaccataagaattaaactatt 240 aatggtgaatagagtttttcactttaacataggcctatcccactggtgggatacgagcca 300 attcgaaagaaaagtcagtcatgtgcttttcagaggatgaaagcttaagataaagactaa 360 aagtgtttgatgctggaggtgggagtggtattatataggtctcagccaagacatgtgata 420 atcactgtagtagtagctggaaagagaaatctgtgactccaattagccagttcctgcaga 480 ccttgtgaggactagaggaagaatgctcctggctgttttgtactgcctgctgtggagttt 540 ccagacctccgctggccatttccctagagcctgtgtctcctctaagaacctgatggagaa 600 ggaatgctgtccaccgtggagcggggacaggagtccctgtggccagctttcaggcagagg660 ttcctgtcagaatatccttctgtccaatgcaccacttgggcctcaatttcccttcacagg720 ggtggatgaccgggagtcgtggccttccgtcttttataataggacctgccagtgctctgg780 caacttcatgggattcaactgtggaaactgcaagtttggcttttggggaccaaactgcac840 agagagacgactcttggtgagaagaaacatcttcgatttgagtgccccagagaaggacaa900 attttttgcctacctcactttagcaaagcataccatcagctcagactatgtcatccccat960 agggacctatggccaaatgaaaaatggatcaacacccatgtttaacgacatcaatattta1020 tgacctctttgtctggatgcattattatgtgtcaatggatgcactgcttgggggatctga1080 aatctggagagacattgattttgcccatgaagcaccagcttttctgccttggcatagact1140 cttcttgttgcggtgggaacaagaaatccagaagctgacaggagatgaaaacttcactat1200 tccatattgggactggcgggatgcagaaaagtgtgacatttgcacagatgagtacatggg1260 aggtcagcaccccacaaatcctaacttactcagcccagcatcattcttctcctcttggca1320 gattgtctgtagccgattggaggagtacaacagccatcagtctttatgcaatggaacgcc1380 ' ' cgagggacctttacggcgtaatcctggaaacca tccagaaccccaaggctccc1440 tgacaaa ctcttcagctgatgtagaattttgcctgagtttgacccaatatgaatctggttccatgga1500 taaagctgccaatttcagctttagaaatacactggaaggatttgctagtccacttactgg1560 gatagcggatgcctctcaaagcagcatgcacaatgccttgcacatctatatgaatggaac1620 , aatgtcccaggtacagggatctgccaacgatcctatcttccttcttcaccatgcatttgt168'0 tgacagtatttttgagcagtggctccgaaggcaccgtcctcttcaagaagtttatccaga1740 agccaatgcacccattggacataaccgggaatcctacatggttccttttataccactgta1800 cagaaatggtgatttctttatttcatccaaagatctgggctatgactatagctatctaca1860 agattcagacccagactcttttcaagactacattaagtcctatttggaacaagcgagtcg1920 gatctggtcatggctccttggggcggcgatggtaggggccgtcctcactgccctgctggc1980 ' agggcttgtgagcttgctgtgtcgtcacaagagaaagcagcttcctgaagaaaagcagcc2040 actcctcatggagaaagaggattaccacagcttgtatcagagccatttataaaaggctta2100 ggcaatagagtagggccaaaaagcctgacctcactctaactcaaagtaatgtccaggttc2160 ccagagaatatctgctggtatttttctgtaaagaccatttgcaaaattgtaacctaatac2220 aaagtgtagccttcttccaactcaggtagaacacacctgtctttgtcttgctgttttcac2280 tcagcccttt taacattttc ccctaagccc atatgtctaa ggaaaggatg ctatttggta 2340 atgaggaact gttatttgta tgtgaattaa agtgctctta tttt 2384 <210> 23 <211> 30 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Probe <900> 23 cctgaggcac ctggaaggag gctgcagtgt 30 <210> 24 <211> 27 <212> DNA
<213> Artificial <220>
<223> artificial sequence is a Probe <400> 24 atcaaaaaac aagcatggcc tcacacc 27 <210> 25 <211> 22 ' <212> DNA
' <213> Artificial <220> , <223> Artificial sequence is a Primer ' <400> 25 ' ctgcttcgct gcatcgctga as 22 <210> 26 <211> 22 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Primer <400> 26 cagactcctc cagtcaggta ca 22 <210> 27 <211> 21 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Primer <400> 27 gcttggtggt taaaacttac c 21 <210> 28 <211> 20 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Primer <400> 28 tgaacagttc tgttggtgta , 20 <210> 29 <211> 27 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Probe <400> 29 ctgcctgcct atgtgacgac aatccgg 27 <210> 30 <211> 20 <212> DNA ' , <213> Artificial ' <220> ' <223> Artificial sequence is a Primer <220>
<221> misc_feature <222> (213)..(213) <223> misc feature is artificial primer <900> 30 caattttggt ggagaacccg 20 <210> 31 <211> 20 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Primer <400> 31 gctgtcggag gtatatggtg 20 <210> 32 <211> 28 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Probe <400> 32 catttcagag agtaacatgg actacaca 28 <210> 33 <211> 21 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Primer <400> 33 gcaagtgcca atgatcagag g , 21 <210> 39 <211> 23 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Primer , <400> 39 atatagactc aggtatacac act 23 <210> 35 <211> 27 <212> DNA
<213> Artificial <220>
<223> Artificial sequence is a Probe <400> 35 tcccatcaga atccaaacaa gaggaag ~ 27

Claims (20)

1. A method of extracting RNA from biological systems comprising contacting a solution containing RNA taken from the system with a substrate containing or to which is affixed a material binding to RNA and withdrawing the RNA by applying negative pressure wherein no centrifugation step exceeds 30 seconds.

2. The method of claim 1 wherein the tissue is homogenized by means of a tissue grinder.

3. The method of claim 1 wherein the biological system is a tissue and the viscosity of the solution containing RNA is reduced prior to contacting the solution with the substrate.

4. The method of claim 1 wherein the negative pressure is from 11.6 to 13.1 psi.

5. A method of extracting RNA from biological systems comprising diluting an RNA
homogenate and contacting a solution containing said diluted RNA homogenate with a substrate containing or to which is affixed a material binding to RNA and withdrawing the RNA by applying negative pressure.

6. The method of claim 5 wherein said RNA homogenate is diluted to 8-2mg of homogenate/diluent.

7. The method of claim 5 wherein no centrifugation step exceeds 30 seconds.

8. A method of extracting RNA from biological systems comprising removing from a sample of such systems, cells without RNA of interest, lysing cells containing RNA, contacting the lysate with a substrate containing or to which is affixed a material to which RNA can bind, applying negative in the absence of centrifugation, and removing RNA bound to the substrate; wherein said method is conducted in less than 6 minutes.

9. The method of claim 8 wherein the cells are lysed with a substance containing guanidinium thiocyanate and the substrate is a silica-gel membrane.

10. The method of claim 8 further comprising the step of washing the lysate.

11. The method of claim further comprising the step of centrifuging the lysate before of after contact with the substrate.

12. A method of extracting RNA from biological systems comprising:
(a) obtaining a sample containing cells from the biological system, (b) removing from the sample, cells without RNA of interest to produce a working sample, (c) lysing the cells containing RNA that is of interest, (d) homogenizing the working sample;
(e) wetting the homogenized working sample, (f) contacting the wetted, homogenized working sample with a substrate containing or to which is affixed a material to which RNA binds to form a substrate-RNA complex, (g) applying negative pressure to the substrate to extract the RNA, (h) washing the extracted RNA
(i) removing water from the substrate-RNA complex, and (j) eluting the RNA
wherein said method is conducted in less than 6 minutes.

13. The method of claim 12 wherein cells without RNA of interest are lysed prior to removal.

14. The method of claim 12 further comprising the step of diluting the homogenized working sample.

15. The method of claim 14 wherein the working sample is diluted to between 8 and 2 mg of homogenate per ml of diluent.

16. The method of claim 15 wherein the working sample is diluted to about 4 mg of homogenate to ml of diluent.

17. The method of claim 14 wherein the diluent comprises a material that preserves RNA.

18. The method of claim 17 wherein the material contains guanidinium thiocyanate.

19. The method of claim 12 further comprising centrifugation steps following steps g, h, i or j and wherein no such centrifugation step exceeds 30 seconds.

20. The method of claim 12 wherein the substrate is a silica-gel membrane.

.'