Systematic Comparison of the T7-IVT and SMART-Based RNA Preamplification Techniques for DNA Microarray Experiments

Abstract

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 TM 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 oligomerspotted 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

Endotoxin-induced gene expression differences in the brain and effects of iNOS inhibition and norepinephrine

“…This becomes obvious especially when studying strongly regulated genes. Additionally, preamplification methods tend to cause a data compression [23][24][25].…”

Single molecule transcription profiling with AFM

“…Genes transcribed at low levels, such as regulatory proteins, exert large biological effects from small changes in expression level [13,18,22]. Considerable work with enzymatic amplification, including PCR-based [23][24][25][26][27][28][29][30][31][32][33], multiple displacement amplification-based (MDA) [24,, and RNA polymerase-based [23,[56][57][58][59][60][61][62][63] protocols, has enabled the use of hybridization microarrays and sequence tag methods to characterize low abundance mRNA samples [64][65][66][67][68][69][70][71][72]. This includes several recent reports of message profiling of single cells [73][74][75][76][77][78][79][80][81][82].…”

Comparison of RNA amplification techniques meeting the demands for the expression profiling of clinical cancer samples

“…This usually takes around 3-4 days to be completed and introduces a new source of bias and results in 5′-truncated transcripts. Degradation of RNA progresses with time and additional rounds of IVT, resulting in 5′-truncated transcripts, as reported in several studies [10,12,16,21,33]. Consequently, GeneChip arrays preferentially contain oligonucleotides derived within 600 bp to the 3′ end of the transcript.…”

“…However, correlation coefficients between methods are usually lower than 0.9. To date, a few studies compared more than one technique to unamplified RNA [5,7,18,20,28,33], all of them chiefly involving time-consuming IVT protocols.…”