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. 2007 Jul 17;35(15):e94. doi: 10.1093/nar/gkm510

Figure 2.

Figure 2.

Experimental procedure utilized for the analysis of 10-year-old matched frozen and formalin-fixed paraffin embedded breast cancer samples. (a) Five micrograms of RNA extracted from the 10-year-old frozen portion of the sample, is reverse-transcribed and the cDNA is double stranded (dsDNA), in four individual reactions. The dsDNA of three reactions undergoes IVT-amplification (MessageAmpII, Ambion), which gives rise to complementary RNA (cRNA) for cDNA microarray analyses. The dsDNA of one reaction is used for PCR experiments. (b) Five micrograms of RNA extracted from the 10-year-old FFPE portion of the sample underwent the exact same process. (c) Single-stranded DNA (ssDNA) obtained by RT of 5 μg of FFPE-RNA is purified and hybridized to the sense-RNA template library. The restored ssDNA is double stranded and purified. Three of the CT-RT reactions undergo IVT-amplification, while the dsDNA of one reaction is used for PCR experiments.