Effective transcriptome amplification for expression profiling on sense-oriented oligonucleotide microarrays.
Gene expression analysis using microarrays of synthetic long oligonucleotides is limited in that it requires substantial amounts of RNA. To obtain these quantities from minute amounts of starting material, protocols were developed that linearly amplify mRNA by cDNA synthesis and in vitro transcription. Since orientation of the product is antisense (aRNA), it is inapplicable for dye-labelling by reverse transcription and hybridization to sense-oriented oligonucleotide arrays. Here, we introduce a novel protocol in which aRNA labelling is achieved by a combination of two reverse and one forward transcription reactions followed by dye-incorporation using Klenow fragment, generating fluorescent antisense cDNA. We demonstrate high fidelity in arrays using up to 10(5)-fold amplification, starting from 2 ng total RNA. The generated data are highly reproducible and maintain relative gene expression levels between samples. These results demonstrate that our protocol describes an efficient and reliable technique to expand the applicability of oligonucleotide arrays to studies where RNA is the limited source material.
Schlingemann
Joerg
J
Division of Molecular Genetics, Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
Thuerigen
Olaf
O
Ittrich
Carina
C
Toedt
Grischa
G
Kramer
Heidi
H
Hahn
Meinhard
M
Lichter
Peter
P
D016373:DNA, Antisense / Q000737:chemistry; D018076:DNA, Complementary / Q000096:biosynthesis; D005456:Fluorescent Dyes / Q000737:chemistry; D020869:Gene Expression Profiling; D006801:Humans; D016014:Linear Models; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D014158:Transcription, Genetic
D016373:DNA, Antisense; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012333:RNA, Messenger
Proc Natl Acad Sci U S A. 1990 Mar;87(5):1663-7
1689846
Science. 2000 Aug 18;289(5482):1197-202
10947988
Genomics. 2003 Oct;82(4):491-7
13679029
Lab Invest. 2004 Jan;84(1):131-7
14647400
Nat Biotechnol. 2002 Apr;20(4):387-92
11923846
Nat Biotechnol. 2002 Sep;20(9):940-3
12172558
Biotechniques. 2002 Oct;33(4):906-12, 914
12398200
Nat Biotechnol. 2000 Apr;18(4):457-9
10748532
Bioinformatics. 2002;18 Suppl 1:S96-104
12169536
Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):3010-4
1557406
Genomics. 2004 Apr;83(4):550-8
15028277
Clin Cancer Res. 2002 May;8(5):1155-66
12006532
Nucleic Acids Res. 2001 Mar 1;29(5):E29
11222780
Nucleic Acids Res. 2002 Feb 15;30(4):e15
11842121
J Mol Diagn. 2003 Feb;5(1):9-14
12552074
Anal Chem. 2002 Apr 15;74(8):1792-7
11985309
J Comput Biol. 2001;8(6):625-37
11747616
Genome Res. 2003 Jul;13(7):1775-85
12805270
Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):392-6
1309614
Nucleic Acids Res. 1999 Mar 15;27(6):1558-60
10037822
Nucleic Acids Res. 2003 Mar 1;31(5):e20
12595569
Nucleic Acids Res. 2003 Jun 15;31(12):e67
12799456
Nat Biotechnol. 2002 Sep;20(9):936-9
12161758
Nat Genet. 2001 Dec;29(4):389-95
11726925
Nat Biotechnol. 2001 Nov;19(11):1011-2
11689839
Nucleic Acids Res. 1992 Apr 11;20(7):1691-6
1579461
Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10614-9
8855227
Physiol Genomics. 2003 Apr 16;13(2):147-56
12700361
Physiol Genomics. 2001 Oct 10;7(1):45-53
11595791