High-accuracy amplification of nanogram total RNA amounts for gene profiling.
Microarray-based gene profiling of laser-assisted microdissected tissues or clinical biopsies is still a challenge since the amount of total RNA in such samples is limited and amplification of RNA is mandatory. Representative amplification of mRNA is highly dependent on the reverse transcription reaction, which is error prone, and on the number of amplification cycles. To improve the accuracy of RNA amplification, we optimized, combined, and tested different amplification strategies for Affymetrix oligonucleotide array hybridization. We demonstrate that different protocols differ significantly in quality of mRNA amplification. To demonstrate the accuracy and reproducibility of our optimized protocol in a clinical setting, we analyzed total RNAs from laser-assisted, microdissected cells of human prostate tissues. On the basis of these results, we recommend a standard reverse transcription reaction for small-sample-transcriptome profiling experiments as part of the Minimal Information about a Microarray Experiment (MIAME) set of standards.
Kenzelmann
Marc
M
Division of Molecular Biology of the Cell I, Deutsches Krebsforschungszentrum Heidelberg, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Klären
Ralf
R
Hergenhahn
Manfred
M
Bonrouhi
Mahnaz
M
Gröne
Hermann-Josef
HJ
Schmid
Wolfgang
W
Schütz
Günther
G
D000818:Animals; D017173:Caenorhabditis elegans; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards*; D016678:Genome; D007834:Lasers; D008297:Male; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D013737:Testis / Q000378:metabolism; D013950:Thymus Gland / Q000166:cytology
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D012313:RNA