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POSTER
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
September 2010
2010
(p.7)
DOI: 10.2144/000113527
Sponsored,vendor-submitted protocol
Sigma Life Science
Sponsored by Sigma-Aldrich
Published in
September 2010
2010
(p.7)
DOI: 10.2144/000113527
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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