( B) Comparison of standard versus overnight Proteinase K digestion. Paired t-test was performed comparing the indicated groups. The Epicentre kit had the highest yield of RNA, and the Roche kit had the lowest yield. The total RNA recovered was diluted to a constant volume before measuring RNA concentration using a fluorometric method. Using each method, RNA was isolated from three 10-Ојm sections from 10 formalin-fixed, paraffin-embedded (FFPE) renal tumors. ( A) Comparison of RNA yields from archival renal tumors using 5 different methods for RNA isolation, including 4 commercial kits. 1 Determination of optimal RNA isolation method for use with archival renal tissue. formalin-fixed paraffin-embedded tissuesДЇig.Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqManВ® qPCR can be optimized by using the MasterPureв„ў RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqManВ® PCR. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Overnight protein digestion resulted in a significant increase in RNA yield. Using the top 3 RNA isolation methods, the manufacturersвЂ™ protocols were modified to include an overnight Proteinase K digestion. However, the difference in RNA yield between the kit from Epicenter and InvitrogenвЂ™s TRIzol method was not significant.

The MasterPureв„ў kit from Epicentre produced the highest RNA yield. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqManВ® PCR.

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