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Nucleic Acids Research Advance Access originally published online on March 30, 2009
Nucleic Acids Research 2009 37(8):e65; doi:10.1093/nar/gkp193
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Nucleic Acids Research, 2009, Vol. 37, No. 8 e65
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Real-time monitoring of aRNA production during T7 amplification to prevent the loss of sample representation during microarray hybridization sample preparation

Isabelle Gilbert1, Sara Scantland1, Isabelle Dufort1, Olga Gordynska2, Aurélie Labbe2, Marc-André Sirard1 and Claude Robert1,*

1Centre de Recherche en Biologie de la Reproduction, Département des sciences animales and 2Département de mathématiques et de statistique, Université Laval, Québec, Canada, G1K 7P4

*To whom correspondence should be addressed. Fax: +1 (418) 656 3766; Email: claude.robert{at}fsaa.ulaval.ca

Received September 25, 2008. Revised March 5, 2009. Accepted March 10, 2009.

Gene expression analysis performed through comparative abundance of transcripts is facing a new challenge with the increasing need to compare samples of known cell number, such as early embryos or laser microbiopsies, where the RNA contents of identical cellular inputs can by nature be variable. When working with scarce tissues, the success of microarray profiling largely depends on the efficiency of the amplification step as determined by its ability to preserve the relative abundance of transcripts in the resulting amplified sample. Maintaining this initial relative abundance across samples is paramount to the generation of physiologically relevant data when comparing samples of different RNA content. The T7 RNA polymerase (T7-IVT) amplification is widely used for microarray sample preparation. Characterization of the reaction's kinetics has clearly indicated that its true linear phase is of short duration and is followed by a nonlinear phase. This second phase leads to modifications in transcript abundance that biases comparison between samples of different types. The impact assessment performed in this study has shown that the standard amplification protocol significantly lowers the quality of microarray data, rendering more than half of differentially expressed candidates undetected and distorting the true proportional differences of all candidates analyzed.


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