Systematic comparison of the T7-IVT and SMART-based RNA preamplification techniques for DNA microarray experiments.
BACKGROUND
Small biological samples obtained from biopsies or laser microdissection often do not yield sufficient RNA for successful microarray hybridization; therefore, RNA amplification is performed before microarray experiments. We compared 2 commonly used techniques for RNA amplification.


METHODS
We compared 2 commercially available methods, Arcturus RiboAmp for in vitro transcription (IVT) and Clontech BD SMART for PCR, to preamplify 50 ng of total RNA isolated from mouse livers and kidneys. Amplification factors of 3 sequences were determined by real-time PCR. Differential expression profiles were compared within and between techniques as well as with unamplified samples with 10K 50mer oligomer-spotted microarrays (MWG Biotech). The microarray results were validated on the transcript and protein levels by comparison with public expression databases.


RESULTS
Amplification factors for specific sequences were lower after 2 rounds of IVT than after 12 cycles of SMART. Furthermore, IVT showed a clear decrease in amplification with increasing distance of the amplified sequences from the polyA tail, indicating generation of smaller products. In the microarray experiments, reproducibility of the duplicates was highest after SMART. In addition, SMART-processed samples showed higher correlation when compared with unamplified samples as well as with expression databases.


CONCLUSIONS
Whenever 1 round of T7-IVT does not yield sufficient product for microarray hybridization, which is usually the case when <200 ng of total RNA is used as starting material, we suggest the use of SMART PCR for preamplification.
Wilhelm
Jochen
J
Department of Pathology, Justus-Liebig-University Giessen, Giessen, Germany. jochen.wilhelm@patho.med.uni-giessen.de
Muyal
Jai Prakash
JP
Best
Johannes
J
Kwapiszewska
Grazyna
G
Stein
Maria Magdalena
MM
Seeger
Werner
W
Bohle
Rainer Maria
RM
Fink
Ludger
L
D020413:3' Untranslated Regions; D000818:Animals; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D011163:Hydroxymethylbilane Synthase / Q000235:genetics; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000737:chemistry* / Q000302:isolation & purification; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D014764:Viral Proteins / Q000737:chemistry*
D020413:3' Untranslated Regions; D011933:Reagent Kits, Diagnostic; D014764:Viral Proteins; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011163:Hydroxymethylbilane Synthase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases