Systematic analysis of T7 RNA polymerase based in vitro linear RNA amplification for use in microarray experiments.
BACKGROUND
The requirement of a large amount of high-quality RNA is a major limiting factor for microarray experiments using biopsies. An average microarray experiment requires 10-100 microg of RNA. However, due to their small size, most biopsies do not yield this amount. Several different approaches for RNA amplification in vitro have been described and applied for microarray studies. In most of these, systematic analyses of the potential bias introduced by the enzymatic modifications are lacking.


RESULTS
We examined the sources of error introduced by the T7 RNA polymerase based RNA amplification method through hybridisation studies on microarrays and performed statistical analysis of the parameters that need to be evaluated prior to routine laboratory use. The results demonstrate that amplification of the RNA has no systematic influence on the outcome of the microarray experiment. Although variations in differential expression between amplified and total RNA hybridisations can be observed, RNA amplification is reproducible, and there is no evidence that it introduces a large systematic bias.


CONCLUSIONS
Our results underline the utility of the T7 based RNA amplification for use in microarray experiments provided that all samples under study are equally treated.
Schneider
Jörg
J
Department of Molecular Genome Analysis, German Cancer Research Centre, Heidelberg 69120, Germany. joerg.schneider@dkfz.de
Buness
Andreas
A
Huber
Wolfgang
W
Volz
Joachim
J
Kioschis
Petra
P
Hafner
Mathias
M
Poustka
Annemarie
A
Sültmann
Holger
H
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology; D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D020224:Expressed Sequence Tags; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012334:RNA, Neoplasm / Q000235:genetics; D015203:Reproducibility of Results; D014764:Viral Proteins
D018076:DNA, Complementary; D012334:RNA, Neoplasm; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases