CA2464691A1 - Methods and systems for dynamic gene expression profiling - Google Patents
Methods and systems for dynamic gene expression profiling Download PDFInfo
- Publication number
- CA2464691A1 CA2464691A1 CA002464691A CA2464691A CA2464691A1 CA 2464691 A1 CA2464691 A1 CA 2464691A1 CA 002464691 A CA002464691 A CA 002464691A CA 2464691 A CA2464691 A CA 2464691A CA 2464691 A1 CA2464691 A1 CA 2464691A1
- Authority
- CA
- Canada
- Prior art keywords
- sample
- oligonucleotide primer
- genes
- specific
- expression profile
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
Abstract
The invention provides compositions, methods and systems for dynamic transcription profiling of two or more samples. The method of the invention comprises the uses of sample-specific primers for cDNA synthesis and for subsequent amplification of the synthesized cDNAs. The levels of abundance o f genes are compared between samples for the identification of differently expressed genes.
Claims (73)
1. A method for comparing gene expression profiles of two or more samples, said method comprising:
(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag is GC rich at its 5' terminal and At rich at its 3'-terminal;
(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;
(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and (d) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.
(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag is GC rich at its 5' terminal and At rich at its 3'-terminal;
(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;
(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and (d) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.
2. The method of claim 1, wherein said step (a) comprises reverse transcribing RNA
from two or more sample sources into first strand cDNA, and wherein said cDNA
is differentially tagged according to their sources.
from two or more sample sources into first strand cDNA, and wherein said cDNA
is differentially tagged according to their sources.
3. The method of claim 1, wherein said plurality of first strand cDNAs is synthesized by reverse transcription using total RNAs or mRNAs derived from said first sample.
4. The method of claim 1, wherein a third oligonucleotide primer comprising said sequence-specific sequence tag of said first oligonucleotide primer is used for said amplifying so as to generate one or more sample-specific amplified products.
5. The method of claim 1, wherein at least one of said two or more samples is derived form the group consisting of: a normal sample, a disease sample, a sample at a given development stage or condition, a sample prior to a given treatment stage or condition, a sample after a given treatment stage or condition, and a sample at a given culturing stage or condition.
6. The method of claim 1, wherein at least one of said two or more samples is derived from the group consisting o~ an animal, an organ, a tissue type, and a cell type.
7. The method of claim 1, wherein said sample-specific sequence in said first oligonucleotide primer is 15-30 nucleotides in length.
8. The method of claim 1, wherein said sample-specific sequence is 20-24 nucleotide in length.
9. The method of claim 1, wherein said first oligonucleotide primer further comprises a sequence of 5' oligo(dT)n VN 3', where n is at least 5; V is dATP, dGTP, or dCTP;
and N is dTTP (or dUTP), dATP, dGTP, or dCTP.
and N is dTTP (or dUTP), dATP, dGTP, or dCTP.
10. The method of claim 1, wherein said first oligonucleotide primer is provided as a mixture of primers comprising [5'-(specific sequence tag)20-24T12-16AN-3', 5'-(specific sequence tag)20-24T12-16CN-3', and 5'-(specific sequence tag)20-24T12-16GN-3'], wherein said specific sequence tags are identical or different for each primer in said mixture.
11. The method of claim 10, wherein n is 12-16.
12. The method of claim 10, wherein in said first oligonucleotide primer, said sample-specific sequence tag is located at the 5' of oligo(dT)n VN.
13. The method of claim 1, further comprising synthesizing one or more second strand cDNAs complementary to said first strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag, wherein step (b) amplifies at least a subset of said second strand cDNAs so as to generate one or more sample-specific amplified products.
14. The method of claim 13, wherein said second oligonucleotide primer further comprises a second sequence which is complementary to a subset of said first strand cDNAs so as to permit the synthesis of one or more second strand cDNAs.
15. The method of claim 14, wherein in said second oligonucleotide primer, said second sequence is located 3' of said first arbitrary sequence.
16. The method of claim 14, wherein said second oligonucleotide further comprises a sequence of (Z)m between said first and second sequences, where Z is a nucleotide which can form base pair with any of A, T, G, or C, and m is at least 2.
17. The method of claim 16, wherein m is 4.
18. The method of claim 14, wherein said second sequence is 5-10 nucleotides in length.
19. The method of claim 18, wherein said second sequence is 6-7 nucleotides in length.
20. The method of claim 13, wherein said first arbitrary sequence within said second oligonucleotide primer is 15-30 nucleotides in length.
21. The method of claim 13, wherein said first arbitrary sequence within said second oligonucleotide primer comprises a A-T rich region and a G-C rich region.
22. The method of claim 21, wherein said G-C rich region is located at 5' of said A-T
rich region.
rich region.
23. The method of claim 13, wherein said second oligonucleotide primer used is the same for said two or more samples to be compared.
24. The method of 4, wherein said amplifying further comprises using a fourth oligonucleotide primer which comprises said first arbitrary sequence tag of said second oligonucleotide primer.
25. The method of claim 24, wherein said fourth oligonucleotide primer used is the same for said two or more samples to be compared.
26. The method of claim 14, wherein said second sequence within said second oligonucleotide primer is gene-family-specific.
27. The method of claim 14, wherein said second sequence within said second oligonucleotide primer is a sequence encoding a peptide specific for a protein family.
28. The method of claim 27, wherein said second sequence comprises a sequence encoding a signature sequence motif for a specific protein family.
29. The method of claim 28, wherein said protein family is selected from the group consisting of: receptor tyrosine kinases, G protein coupled receptors, seven transmembrane receptors, ion channels, cytokine receptors, tumor markers, MAPK cascade kinases, transcriptional factors, GTPases, ATPases, and development protein markers.
30. The method of claim 1, wherein said first strand cDNA is synthesized in a solution without attaching to a solid support.
31. The method of claim 1, wherein said first strand cDNA is synthesized attaching to a solid support.
32. The method of claim 31, wherein said solid support is a microparticle or an inner wall of a reaction tube.
33. The method of claim 13, wherein said method further comprises separating said one or more second strand cDNA from said plurality of first strand cDNA before amplifying said one or more second strand cDNAs.
34. The method of claim 4, wherein said third oligonucleotide primer is linked to a detectable label.
35. The method of claim 34, wherein said detectable label is selected from a group consisting of: fluorescent labels, radioactive labels, colorimetrical labels, magnetic labels, and enzymatic labels.
36. The method of claim 35, wherein said detectable label is a fluorescent label.
37. The method of claim 34, wherein said third oligonucleotide primer used for each of said two or more samples is labeled with a sample-specific label.
38. The method of claim 1, wherein said one or more amplified products are sampled at a predetermined time or cycle interval during the amplification.
39. The method of claim 38, wherein the abundance is detected for each sampled amplified product.
40. The method of 1, wherein said method further comprises separating said one or more amplified products before detecting the abundance of said one or more amplified products.
41. The method of claim 40, wherein said one or more amplified products are separated and their abundance detected by chromatography.
42 The method of claim 40, wherein said one or more amplified products are separated and their abundance detected by measurement of fluorescence.
43. The method of claim 40, wherein said one or more amplified products are separated and their abundance detected by measurement of optical density.
44. The method of claim 40, wherein said one or more amplified products are separated and their abundance detected by mass spectrometry.
45. The method of claim 40, wherein said one or more amplified products are separated by electrophoresis.
46. The method of claim 45, wherein said one or more amplified products are separated by capillary electrophoresis.
47. The method of claim 1, wherein said difference in the expression profile of said one or more genes is measured by a ratio of sample-specific detectable labels on amplified products from said genes between two or more samples.
48. The method of claim 1, wherein said method further comprises generating an amplification plot, calculating a Ct of amplification for each of said one or more genes, and measuring the difference in the expression profile by a ratio of said Cts.
49. The method of claim 1, wherein said method further comprises collecting one or more genes which are differentially expressed and identifying the sequence identities of said one or more genes by DNA sequencing.
50. The method of claim 1, wherein said amplifying is performed by PCR.
51. A method for comparing gene expression profiles of two or more samples, said method comprising:
(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said first oligonucleotide primer comprises at least one degenerate nucleotide;
(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;
(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and (d) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.
(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said first oligonucleotide primer comprises at least one degenerate nucleotide;
(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;
(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and (d) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.
52. A method for comparing gene expression profiles of two or more samples, said method comprising:
(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag comprises at least one artificial nucleotide;
(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;
(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and (d) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.
(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag comprises at least one artificial nucleotide;
(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;
(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and (d) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.
53. A method for comparing gene expression profiles of two or more samples, said method comprising:
(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag is GC rich at its 5' terminal and AT rich at its 3' terminal;
(b) selectively synthesizing one or more second strand cDNAs complementary to said first strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;
(c) amplifying said one or more second strand cDNA so as to generate one or more sample-specific amplified products;
(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and (e) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.
(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag is GC rich at its 5' terminal and AT rich at its 3' terminal;
(b) selectively synthesizing one or more second strand cDNAs complementary to said first strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;
(c) amplifying said one or more second strand cDNA so as to generate one or more sample-specific amplified products;
(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and (e) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.
54. A method for comparing gene expression profiles of two or more samples, said method comprising:
(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said first oligonucleotide primer comprises at least one degenerate nucleotide;
(b) selectively synthesizing one or more second strand cDNAs complementary to said first strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;
(c) amplifying said one or more second strand cDNA so as to generate one or more sample-specific amplified products;
(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and (e) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.
(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said first oligonucleotide primer comprises at least one degenerate nucleotide;
(b) selectively synthesizing one or more second strand cDNAs complementary to said first strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;
(c) amplifying said one or more second strand cDNA so as to generate one or more sample-specific amplified products;
(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and (e) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.
55. A method for comparing gene expression profiles of two or more samples, said method comprising:
(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag comprises at least one artificial nucleotide;
(b) selectively synthesizing one or more second strand cDNAs complementary to said first strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;
(c) amplifying said one or more second strand cDNA so as to generate one or more sample-specific amplified products;
(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and (e) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.
(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag comprises at least one artificial nucleotide;
(b) selectively synthesizing one or more second strand cDNAs complementary to said first strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;
(c) amplifying said one or more second strand cDNA so as to generate one or more sample-specific amplified products;
(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and (e) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.
56. A method of identifying a modulator which regulates one or more gene expression in a sample, said method comprising:
(a) synthesizing a plurality of first strand cDNAs, before contacting said sample with said modulator, using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag is GC rich at its 5' terminal and At rich at its 3' terminal;
(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;
(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said sample; and (d) comparing the expression profile of said one or more genes in said sample before contacting with said modulator with an expression profile of said one or more genes in said sample after contacting said modulator, wherein a difference in the expression profile indicates said modulator regulating one or more gene expression in said sample.
(a) synthesizing a plurality of first strand cDNAs, before contacting said sample with said modulator, using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag is GC rich at its 5' terminal and At rich at its 3' terminal;
(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;
(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said sample; and (d) comparing the expression profile of said one or more genes in said sample before contacting with said modulator with an expression profile of said one or more genes in said sample after contacting said modulator, wherein a difference in the expression profile indicates said modulator regulating one or more gene expression in said sample.
57. A method of identifying a modulator which regulates one or more gene expression in a sample, said method comprising:
(a) synthesizing a plurality of first strand cDNAs, before contacting said sample with said modulator, using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said first oligonucleotide primer comprises at least one degenerate nucleotide;
(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;
(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said sample; and (d) comparing the expression profile of said one or more genes in said sample before contacting with said modulator with an expression profile of said one or more genes in said sample after contacting said modulator, wherein a difference in the expression profile indicates said modulator regulating one or more gene expression in said sample.
(a) synthesizing a plurality of first strand cDNAs, before contacting said sample with said modulator, using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said first oligonucleotide primer comprises at least one degenerate nucleotide;
(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;
(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said sample; and (d) comparing the expression profile of said one or more genes in said sample before contacting with said modulator with an expression profile of said one or more genes in said sample after contacting said modulator, wherein a difference in the expression profile indicates said modulator regulating one or more gene expression in said sample.
58. A method of identifying a modulator which regulates one or more gene expression in a sample, said method comprising:
(a) synthesizing a plurality of first strand cDNAs, before contacting said sample with said modulator, using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag is GC rich at its 5' terminal and At rich at its 3' terminal;
(b) synthesizing one or more second strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;
(c) amplifying said second strand cDNAs so as to generate one or more sample-specific amplified products;
(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said sample; and (e) comparing the expression profile of said one or more genes in said sample before contacting with said modulator with an expression profile of said one or more genes in said sample after contacting said modulator, wherein a difference in the expression profile indicates said modulator regulating one or more gene expression in said sample.
(a) synthesizing a plurality of first strand cDNAs, before contacting said sample with said modulator, using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag is GC rich at its 5' terminal and At rich at its 3' terminal;
(b) synthesizing one or more second strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;
(c) amplifying said second strand cDNAs so as to generate one or more sample-specific amplified products;
(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said sample; and (e) comparing the expression profile of said one or more genes in said sample before contacting with said modulator with an expression profile of said one or more genes in said sample after contacting said modulator, wherein a difference in the expression profile indicates said modulator regulating one or more gene expression in said sample.
59. A method of identifying a modulator which regulates one or more gene expression in a sample, said method comprising:
(a) synthesizing a plurality of first strand cDNAs, before contacting said sample with said modulator, using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said first oligonucleotide primer comprises at least one degenerate nucleotide;
(b) synthesizing one or more second strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;
(c) amplifying said second strand cDNAs so as to generate one or more sample-specific amplified products;
(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said sample; and (e) comparing the expression profile of said one or more genes in said sample before contacting with said modulator with an expression profile of said one or more genes in said sample after contacting said modulator, wherein a difference in the expression profile indicates said modulator regulating one or more gene expression in said sample.
(a) synthesizing a plurality of first strand cDNAs, before contacting said sample with said modulator, using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said first oligonucleotide primer comprises at least one degenerate nucleotide;
(b) synthesizing one or more second strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;
(c) amplifying said second strand cDNAs so as to generate one or more sample-specific amplified products;
(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said sample; and (e) comparing the expression profile of said one or more genes in said sample before contacting with said modulator with an expression profile of said one or more genes in said sample after contacting said modulator, wherein a difference in the expression profile indicates said modulator regulating one or more gene expression in said sample.
60. A composition for detecting the level of gene expression, comprising a first oligonucleotide primer, wherein said first oligonucleotide primer comprises a sample-specific sequence tag and wherein said sample-specific sequence tag is GC rich at its 5' terminal and AT
rich at its 3' terminal.
rich at its 3' terminal.
61. The composition of claim 60, further comprising a second oligonucleotide primer which comprises a first arbitrary sequence tag.
62. The composition of claim 61, further comprising a third oligonucleotide primer comprising said sequence-specific sequence tag of said first oligonucleotide primer.
63. The composition of claim 62, further comprising a fourth oligonucleotide primer which comprises said first arbitrary sequence tag.
64. The composition of claim 60, wherein said second primer further comprises a second sequence which is complementary to a sequence of said first strand cDNA.
65. The composition of claim 60, further comprising one or more components selected from the group of: a reverse transcriptase, a DNA polymerase, a reaction buffer for said reverse transcriptase, a reaction buffer for said DNA polymerase, and dNTPs.
66. A composition for detecting the level of gene expression, comprising a first oligonucleotide primer, wherein said first oligonucleotide primer comprises a sample-specific sequence tag and wherein said first oligonucleotide primer comprises at least one degenerate nucleotide.
67. A kit for detecting the level of gene expression, comprising a first oligonucleotide primer, wherein said first oligonucleotide primer comprises a sample-specific sequence tag and wherein said sample-specific sequence tag is GC rich at its 5' terminal and AT
rich at its 3' terminal, and packaging material thereof.
rich at its 3' terminal, and packaging material thereof.
68. The kit of claim 67, further comprising a second oligonucleotide primer which comprises a first arbitrary sequence tag.
69. The kit of claim 67, further comprising a third oligonucleotide primer comprising said sequence-specific sequence tag of said first oligonucleotide primer.
70. The kit of claim 68, further comprising a fourth oligonucleotide primer which comprises said first arbitrary sequence tag.
71. The composition of claim 68, wherein said second primer further comprises a second sequence which is complementary to a sequence of said first strand cDNA.
72. The composition of claim 67, further comprising one or more components selected from the group of: a reverse transcriptase, a DNA polymerase, a reaction buffer for said reverse transcriptase, a reaction buffer for said DNA polymerase, and dNTPs.
73. A kit for detecting the level of gene expression, comprising a first oligonucleotide primer, wherein said first oligonucleotide primer comprises a sample-specific sequence tag and wherein said first oligonucleotide primer comprises at least one degenerate nucleotide, and packaging material thereof.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US34614001P | 2001-10-24 | 2001-10-24 | |
US60/346,140 | 2001-10-24 | ||
US10/113,034 | 2002-04-01 | ||
US10/113,034 US20030077611A1 (en) | 2001-10-24 | 2002-04-01 | Methods and systems for dynamic gene expression profiling |
PCT/US2002/034056 WO2003035841A2 (en) | 2001-10-24 | 2002-10-24 | Methods and systems for dynamic gene expression profiling |
Publications (2)
Publication Number | Publication Date |
---|---|
CA2464691A1 true CA2464691A1 (en) | 2003-05-01 |
CA2464691C CA2464691C (en) | 2013-03-12 |
Family
ID=26810649
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2464691A Expired - Fee Related CA2464691C (en) | 2001-10-24 | 2002-10-24 | Methods and systems for dynamic gene expression profiling |
Country Status (7)
Country | Link |
---|---|
US (2) | US20030077611A1 (en) |
EP (1) | EP1490511A4 (en) |
JP (2) | JP5068417B2 (en) |
KR (2) | KR101045225B1 (en) |
AU (2) | AU2002349908B2 (en) |
CA (1) | CA2464691C (en) |
WO (1) | WO2003035841A2 (en) |
Families Citing this family (63)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030077611A1 (en) | 2001-10-24 | 2003-04-24 | Sention | Methods and systems for dynamic gene expression profiling |
US7445893B2 (en) | 2002-04-12 | 2008-11-04 | Primera Biosystems, Inc. | Sampling method for amplification reaction analysis |
US7081339B2 (en) * | 2002-04-12 | 2006-07-25 | Primera Biosystems, Inc. | Methods for variation detection |
WO2004048528A2 (en) * | 2002-11-21 | 2004-06-10 | Primera Biosystems | Sampling method and apparatus for amplification reaction analysis |
US20040236603A1 (en) * | 2003-05-22 | 2004-11-25 | Biospect, Inc. | System of analyzing complex mixtures of biological and other fluids to identify biological state information |
US7425700B2 (en) * | 2003-05-22 | 2008-09-16 | Stults John T | Systems and methods for discovery and analysis of markers |
AU2005220746A1 (en) * | 2004-03-05 | 2005-09-22 | Medical College Of Ohio | Methods and compositions for assessing nucleic acids and alleles |
EP1745157A4 (en) * | 2004-04-12 | 2008-06-11 | Ohio Med College | Methods and compositions for assaying analytes |
US20050244973A1 (en) * | 2004-04-29 | 2005-11-03 | Predicant Biosciences, Inc. | Biological patterns for diagnosis and treatment of cancer |
US7598080B2 (en) | 2004-08-20 | 2009-10-06 | Carl Deirmengian | Diagnostic assay for source of inflammation |
US20070033062A1 (en) * | 2005-04-28 | 2007-02-08 | Oligoengine, Inc. | Business methods, systems, and computer programs for designing customized biological assays and producing related catalogs and kits |
US7368246B2 (en) * | 2005-09-15 | 2008-05-06 | Primera Biosystems, Inc. | Quantitative gene expression profiling |
AT502823B1 (en) * | 2005-11-29 | 2007-06-15 | Seitz Alexander Dr | POLYNUCLEOTIDE AMPLIFICATION |
WO2008093098A2 (en) | 2007-02-02 | 2008-08-07 | Illumina Cambridge Limited | Methods for indexing samples and sequencing multiple nucleotide templates |
EP2147118A4 (en) * | 2007-04-27 | 2010-09-01 | Eragen Biosciences Inc | Materials and methods for detection of hepatitis c virus |
EP2326732A4 (en) * | 2008-08-26 | 2012-11-14 | Fluidigm Corp | Assay methods for increased throughput of samples and/or targets |
GB0901593D0 (en) | 2009-01-30 | 2009-03-11 | Touchlight Genetics Ltd | Production of closed linear DNA |
ES2555389T3 (en) | 2009-03-30 | 2015-12-30 | Illumina, Inc. | Analysis of gene expression in individual cells |
CN103952482A (en) | 2009-04-02 | 2014-07-30 | 弗卢伊蒂格姆公司 | Multi-primer amplification method for barcoding of target nucleic acids |
GB201013153D0 (en) | 2010-08-04 | 2010-09-22 | Touchlight Genetics Ltd | Primer for production of closed linear DNA |
WO2012129363A2 (en) * | 2011-03-24 | 2012-09-27 | President And Fellows Of Harvard College | Single cell nucleic acid detection and analysis |
SG10201605049QA (en) | 2011-05-20 | 2016-07-28 | Fluidigm Corp | Nucleic acid encoding reactions |
US9428799B2 (en) | 2011-07-25 | 2016-08-30 | Bioinventors & Entrepreneurs Network Llc | Method for determining an allele profile of nucleic acid |
US8932989B2 (en) | 2011-07-25 | 2015-01-13 | Bioinventors & Entrepreneurs Network, Llc | Sieving of nucleic acid samples |
US9133567B2 (en) | 2011-07-25 | 2015-09-15 | Bioinventors & Entrepreneurs Network Llc | Method for determining an attribute profile of biological samples |
EP2807271B1 (en) | 2012-01-24 | 2018-08-22 | CD Diagnostics, Inc. | System for detecting infection in synovial fluid |
JP6375230B2 (en) | 2012-02-27 | 2018-08-15 | セルラー リサーチ, インコーポレイテッド | Compositions and kits for molecular counting |
EP2852682B1 (en) | 2012-05-21 | 2017-10-04 | Fluidigm Corporation | Single-particle analysis of particle populations |
ES2917400T3 (en) * | 2012-07-26 | 2022-07-08 | Illumina Inc | Compositions and methods for nucleic acid amplification |
JP5997278B2 (en) * | 2012-07-30 | 2016-09-28 | 株式会社日立製作所 | Two-dimensional cDNA library device with tag sequence, gene expression analysis method and gene expression analysis apparatus using the same |
WO2014039963A1 (en) * | 2012-09-10 | 2014-03-13 | Biofire Diagnostics, Inc. | Multiple amplification cycle detection |
WO2014108850A2 (en) * | 2013-01-09 | 2014-07-17 | Yeda Research And Development Co. Ltd. | High throughput transcriptome analysis |
US10030240B2 (en) * | 2013-03-12 | 2018-07-24 | Hitachi, Ltd. | Two-dimensional cell array device and apparatus for gene quantification and sequence analysis |
KR20230074639A (en) | 2013-08-28 | 2023-05-30 | 벡톤 디킨슨 앤드 컴퍼니 | Massively parallel single cell analysis |
GB201415789D0 (en) | 2014-09-05 | 2014-10-22 | Touchlight Genetics Ltd | Synthesis of DNA |
WO2016138496A1 (en) | 2015-02-27 | 2016-09-01 | Cellular Research, Inc. | Spatially addressable molecular barcoding |
US11535882B2 (en) | 2015-03-30 | 2022-12-27 | Becton, Dickinson And Company | Methods and compositions for combinatorial barcoding |
WO2016162997A1 (en) * | 2015-04-09 | 2016-10-13 | 株式会社日立製作所 | Gene analysis system |
CN107580632B (en) | 2015-04-23 | 2021-12-28 | 贝克顿迪金森公司 | Methods and compositions for whole transcriptome amplification |
JP6940484B2 (en) | 2015-09-11 | 2021-09-29 | セルラー リサーチ, インコーポレイテッド | Methods and compositions for library normalization |
EP3390658B1 (en) | 2015-12-16 | 2022-08-03 | Standard BioTools Inc. | High-level multiplex amplification |
US10301677B2 (en) | 2016-05-25 | 2019-05-28 | Cellular Research, Inc. | Normalization of nucleic acid libraries |
US10202641B2 (en) | 2016-05-31 | 2019-02-12 | Cellular Research, Inc. | Error correction in amplification of samples |
US10640763B2 (en) | 2016-05-31 | 2020-05-05 | Cellular Research, Inc. | Molecular indexing of internal sequences |
KR102363716B1 (en) | 2016-09-26 | 2022-02-18 | 셀룰러 리서치, 인크. | Determination of protein expression using reagents having barcoded oligonucleotide sequences |
JP6244439B2 (en) * | 2016-11-22 | 2017-12-06 | 株式会社日立製作所 | Two-dimensional cell array device and apparatus for gene quantification and sequence analysis |
US11319583B2 (en) * | 2017-02-01 | 2022-05-03 | Becton, Dickinson And Company | Selective amplification using blocking oligonucleotides |
EP4345172A2 (en) | 2017-06-05 | 2024-04-03 | Becton, Dickinson and Company | Sample indexing for single cells |
KR20190085429A (en) | 2018-01-10 | 2019-07-18 | 두산공작기계 주식회사 | Apparatus and method for compensating spindle of machine tool |
US11365409B2 (en) | 2018-05-03 | 2022-06-21 | Becton, Dickinson And Company | Molecular barcoding on opposite transcript ends |
CN112272710A (en) | 2018-05-03 | 2021-01-26 | 贝克顿迪金森公司 | High throughput omics sample analysis |
EP3861134A1 (en) | 2018-10-01 | 2021-08-11 | Becton, Dickinson and Company | Determining 5' transcript sequences |
US11932849B2 (en) | 2018-11-08 | 2024-03-19 | Becton, Dickinson And Company | Whole transcriptome analysis of single cells using random priming |
WO2020123384A1 (en) | 2018-12-13 | 2020-06-18 | Cellular Research, Inc. | Selective extension in single cell whole transcriptome analysis |
EP3914728B1 (en) | 2019-01-23 | 2023-04-05 | Becton, Dickinson and Company | Oligonucleotides associated with antibodies |
WO2021016239A1 (en) | 2019-07-22 | 2021-01-28 | Becton, Dickinson And Company | Single cell chromatin immunoprecipitation sequencing assay |
AU2020326698A1 (en) | 2019-08-05 | 2022-02-24 | Seer, Inc. | Systems and methods for sample preparation, data generation, and protein corona analysis |
CN114729350A (en) | 2019-11-08 | 2022-07-08 | 贝克顿迪金森公司 | Obtaining full-length V (D) J information for immunohistorian sequencing using random priming |
WO2021146207A1 (en) | 2020-01-13 | 2021-07-22 | Becton, Dickinson And Company | Methods and compositions for quantitation of proteins and rna |
WO2021231779A1 (en) | 2020-05-14 | 2021-11-18 | Becton, Dickinson And Company | Primers for immune repertoire profiling |
US11932901B2 (en) | 2020-07-13 | 2024-03-19 | Becton, Dickinson And Company | Target enrichment using nucleic acid probes for scRNAseq |
US11739443B2 (en) | 2020-11-20 | 2023-08-29 | Becton, Dickinson And Company | Profiling of highly expressed and lowly expressed proteins |
WO2023235685A1 (en) * | 2022-06-01 | 2023-12-07 | Bio-Rad Laboratories, Inc. | Method and system for detecting reverse transcriptase activity by digital assay |
Family Cites Families (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE198773T1 (en) * | 1991-10-23 | 2001-02-15 | Baylor College Medicine | FINGERPRINT IDENTIFICATION OF BACTERIAL STRAINS USING AMPLIFICATION OF REPETITIVE DNA SEQUENCES |
US6200747B1 (en) * | 1992-01-28 | 2001-03-13 | North Shore University Hospital Research Corp. | Method and kits for detection of fragile X specific, GC-rich DNA sequences |
US6110680A (en) * | 1993-11-12 | 2000-08-29 | The Scripps Research Institute | Method for simultaneous identification of differentially expressed mRNAs and measurement of relative concentrations |
US5710628A (en) | 1994-12-12 | 1998-01-20 | Visible Genetics Inc. | Automated electrophoresis and fluorescence detection apparatus and method |
AU2264197A (en) * | 1996-02-09 | 1997-08-28 | Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The | Restriction display (rd-pcr) of differentially expressed mrnas |
JP4000605B2 (en) | 1996-07-24 | 2007-10-31 | 株式会社日立製作所 | DNA sample preparation device and electrophoretic analyzer using the same |
CA2278465C (en) | 1997-01-30 | 2010-04-27 | University Technology Corporation | Diagnosis and treatment of myocardial failure |
WO1999014368A2 (en) | 1997-09-15 | 1999-03-25 | Whitehead Institute For Biomedical Research | Methods and apparatus for processing a sample of biomolecular analyte using a microfabricated device |
DE19822287C2 (en) * | 1998-05-18 | 2003-04-24 | Switch Biotech Ag | Cloning vector, its production and use for the analysis of mRNA expression patterns |
DE19840731A1 (en) * | 1998-09-07 | 2000-03-09 | Hoechst Marion Roussel De Gmbh | Two-color differential display as a method for the detection of regulated genes |
US7115365B2 (en) | 1998-12-07 | 2006-10-03 | Olympus Optical Co., Ltd. | Method of analyzing a target nucleic acid |
WO2000035274A1 (en) * | 1998-12-17 | 2000-06-22 | The Johns Hopkins University School Of Medicine | Olfactory receptor expression libraries and methods of making and using them |
AU4672400A (en) | 1999-04-29 | 2000-11-17 | Genome Therapeutics Corporation | Device for rapid dna sample processing with integrated liquid handling, thermocycling, and purification |
DE19921419A1 (en) * | 1999-05-08 | 2000-11-16 | Univ Ruprecht Karls Heidelberg | Method for the specific detection and identification of retroviral nucleic acids / retroviruses in a specimen |
US6180349B1 (en) * | 1999-05-18 | 2001-01-30 | The Regents Of The University Of California | Quantitative PCR method to enumerate DNA copy number |
DE19963857A1 (en) * | 1999-12-30 | 2001-07-26 | Qiagen Gmbh | Primers, in particular for primer-dependent nucleic acid synthesis processes and nucleic acid amplification processes |
EP1257664A4 (en) | 2000-01-28 | 2006-04-05 | Althea Technologies Inc | Methods for analysis of gene expression |
US6706476B1 (en) * | 2000-08-22 | 2004-03-16 | Azign Bioscience A/S | Process for amplifying and labeling single stranded cDNA by 5′ ligated adaptor mediated amplification |
AU2001286784A1 (en) | 2000-08-25 | 2002-03-04 | Genome Therapeutics Corporation | Device for rapid dna sample processing with integrated liquid handling, thermocycling, and purification |
US6482615B2 (en) | 2001-03-02 | 2002-11-19 | Integrated Genetic Devices Ltd. | Method and apparatus for effecting rapid thermal cycling of samples in microtiter plate size |
US20030077611A1 (en) | 2001-10-24 | 2003-04-24 | Sention | Methods and systems for dynamic gene expression profiling |
WO2004048528A2 (en) | 2002-11-21 | 2004-06-10 | Primera Biosystems | Sampling method and apparatus for amplification reaction analysis |
-
2002
- 2002-04-01 US US10/113,034 patent/US20030077611A1/en not_active Abandoned
- 2002-10-24 AU AU2002349908A patent/AU2002349908B2/en not_active Ceased
- 2002-10-24 KR KR1020047006047A patent/KR101045225B1/en not_active IP Right Cessation
- 2002-10-24 JP JP2003538342A patent/JP5068417B2/en not_active Expired - Fee Related
- 2002-10-24 WO PCT/US2002/034056 patent/WO2003035841A2/en active Application Filing
- 2002-10-24 EP EP02786493A patent/EP1490511A4/en not_active Withdrawn
- 2002-10-24 CA CA2464691A patent/CA2464691C/en not_active Expired - Fee Related
- 2002-10-24 KR KR1020117004003A patent/KR20110026024A/en not_active Application Discontinuation
-
2005
- 2005-12-23 US US11/318,278 patent/US7550266B2/en not_active Expired - Fee Related
-
2008
- 2008-09-26 AU AU2008229652A patent/AU2008229652B2/en not_active Ceased
-
2009
- 2009-10-14 JP JP2009237655A patent/JP4951049B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
KR20110026024A (en) | 2011-03-14 |
EP1490511A4 (en) | 2007-06-06 |
EP1490511A2 (en) | 2004-12-29 |
AU2002349908B2 (en) | 2008-06-26 |
US20030077611A1 (en) | 2003-04-24 |
KR20040068125A (en) | 2004-07-30 |
CA2464691C (en) | 2013-03-12 |
US7550266B2 (en) | 2009-06-23 |
JP5068417B2 (en) | 2012-11-07 |
AU2008229652B2 (en) | 2012-07-26 |
US20060105380A1 (en) | 2006-05-18 |
KR101045225B1 (en) | 2011-06-30 |
JP4951049B2 (en) | 2012-06-13 |
WO2003035841A2 (en) | 2003-05-01 |
AU2008229652A1 (en) | 2008-10-23 |
JP2010046073A (en) | 2010-03-04 |
WO2003035841A3 (en) | 2004-10-14 |
JP2005507661A (en) | 2005-03-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2464691A1 (en) | Methods and systems for dynamic gene expression profiling | |
Li et al. | MicroRNA detection by microarray | |
JP5118056B2 (en) | Polynucleotide amplification | |
US20030175908A1 (en) | Methods and means for manipulating nucleic acid | |
WO2002027029A2 (en) | Method for determining relative abundance of nucleic acid sequences | |
KR20010033992A (en) | Solid phase selection of differentially expressed genes | |
CN106834472A (en) | BCR diversity detection kit and application | |
CN110157785A (en) | A kind of unicellular RNA sequencing library construction method | |
US20150354000A1 (en) | Method of analysis of composition of nucleic acid mixtures | |
EP2510114B1 (en) | Rna analytics method | |
Luo et al. | Emergence of bias during the synthesis and amplification of cDNA for scRNA-seq | |
US20220177964A1 (en) | A high throughput sequencing method and kit | |
EP1536022A1 (en) | Method for comparing gene expression level | |
Zhao et al. | 3′-end cDNA pool suitable for differential display from a small number of cells | |
US20030143593A1 (en) | Quantative method for measuring gene expression | |
US20080286766A1 (en) | Comparing Method for Expression Amount of the Same Gene from Different Sources by Base Sequence Measurement | |
US20030207269A9 (en) | Preferential display | |
Freeman | Differential Display of Gene Expression | |
Brown et al. | RNA sequencing with next-generation sequencing | |
US20040142339A1 (en) | Methods for analysis of rna | |
CN100460520C (en) | Comparation of expression account of the same gene in different original resources by base sequence determination | |
Russo et al. | RNA Sequencing in the Human Breast | |
EP3798318A1 (en) | A high throughput sequencing method and kit | |
Spira | Current methods of gene expression analysis and quantification | |
Santucci-Pereira et al. | RNA Sequencing in the Human Breast |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
MKLA | Lapsed |
Effective date: 20161024 |
|
MKLA | Lapsed |
Effective date: 20161024 |