The increasing need tobroaden the use of microarray technology has resulted in approaches toamplify the target RNA. Global amplification of RNA has been developedand used in gene expression analysis before the microarray techniqueappeared (Eberwine et al ., 1990). In this method, mRNA is reversetranscribed with an oligo dT/T7 promoter primer. After synthesis ofdouble stranded cDNA, antisense RNA is transcribed in vitro generallyresulting in a 1000-fold amplification of the original amount. Ifnecessary, a second round of amplification can be performed simply byrepeating the procedure. The second round is primed with randomhexameres, resulting in aRNA with reduced transcript lengths. Dataresults are not affected as long as probes on the microarrays also are3’ biased.

Atthe present this approach appears to be the most promising in regardsto gene expression analysis on material derived from sensitiveselection techniques such as immunomagnetic bead selection (IMS) andlaser capture micro dissection (LCM).

We have adopted 2 roundsof amplification as a standard and follow the protocol described byBaugh et al., (2002) with some minor enzyme modifications.

GlobalmRNA amplification has been applied in several publications, howeverthere has been minimal quantitative documentation of the linearity ofthe amplification procedure. We performed statistical analysis(multiple hypothesis testing and ANOVA variance analysis) on a data setgenerated from amplified and non-amplified material. We found a valueof less than 10% false discovery rate of significantly differentiallyexpressed genes when comparing the two data sets (amplified vs.non-amplified). From the ANOVA, we concluded that the variance due toamplification was relatively low compared to the overall variance. Forfurther reading see Nygaard et al., (2003) BMC Genomics.





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