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TransPlex® WTA2 Whole Transcriptome Amplification of RNA from Low-Cell Number Samples
Ken Heuerman and Brian Ward
Sigma Life Science
Sponsored by Sigma-Aldrich
Published in December 2010 2010 (p.8) DOI:  10.2144/000113594 Sponsored,vendor-submitted protocol
The efficacy of amplification of small quantities of total RNA with the Transplex® Complete Whole Transcriptome Amplification Kit (WTA2) was examined in this study. Total RNA extracted from decreasing numbers of FACS-isolated bone marrow stem cells(10-,100-,and1000-cell samples) was amplified with the Transplex® Complete WTA2 Kit. A call rate of 58.8% of unique biological array features was observed for the 10-cellvs.100-cell microarray analysis, with a similar call rate of 61.46% for 10-cells vs.1000-cells. Greater than 90% commonality existed between the intersecting data sets for the two analyses. After a more stringent screening (p=0.0001), the10-cell vs.100-cell comparison revealed 5568 intersecting features. The comparable analysis for 10 cells vs.1000 cells resulted in 4977 features, with 3862 features common to both comparisons. In addition, the effect of decreasing RNA input on amplification efficiency was examined. Results indicated that an adjustment to the library synthesis primer concentration allowed for maintenance of linear amplification and representative qPCR at low RNA input quantities. This adjustment proved to be critical for downstream qPCR applications, but not for the case where the amplification product was used as microarray target. This study confirms that the Transplex® Complete Whole Transcriptome Amplification Kit can effectively amplify low input quantities of RNA, approaching the single cell level.
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