Patent ID: 7960147

Claim:
A method for determining Thymidylate Synthase (TS) expression in a fixed paraffin-embedded tissue sample using precipitation and reverse transcription, comprising: (a) deparaffinizing the tissue sample to obtain a deparaffinized sample; (b) prior to precipitation and reverse transcription, efficiently isolating RNA from the deparaffinized sample by heating the deparaffinized sample in a solution comprising an effective concentration of a chaotropic agent to a temperature in the range of 75 to about 100° C. for a time period of about 5 to about 120 minutes and then recovering said RNA from said solution; (c) subjecting the isolated RNA to RT-PCR amplification using a pair of oligonucleotide primers capable of amplifying a region of the TS gene mRNA to obtain an amplified sample; and (d) determining the quantity of TS mRNA relative to the quantity of an internal control β-actin gene mRNA, wherein the method provides quantitation of TS mRNA relative to the internal control gene mRNA, in RNA isolated from any of a range of tissue and tumor types; wherein the heating step increases the amount of cDNA that is amplified in the RT-PCR amplification step from 3 to 1000 fold in comparison to a deparaffinized tissue sample that is subject to similar RNA isolation conditions as in step (b) but not heated.