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A PCR aided transcript titration assay to quantify mRNA levels in cellular RNA
Abstracts
111
A PCR AIDED TRANSCRIPT TITRATION ASSAY TO QUANTIFY mRNA LEVELS IN CELLULAR RNA. Apke van den Bera (1), Klaas Kok (1), Egbert Smit (2), Charles H.C.M. Buys (1) Departments of Medical Genetics (1) and Pulmonology (2), University of Groningen, Groningen, The Netherlands. We developed a general method to quantify the amount of a specific mRNA in a given amount of total cellular RNA. The method involves the construction of a recombinant cDNA clone by ligating a small restriction fragment into a unique restriction site of the cellular cDNA clone. Two primers were selected on either site of this restriction site. Upon PCR analysis, the recombinant cDNA gives rise to a slightly longer PCR product than the cellular cDNA. Recombinant RNA, to be used as an internal standard, was synthesized in vitro, using the recombinant cDNA clone as a template. Samples containing a fixed amount of cellular RNA and varying amounts of recombinant RNA are reverse transcribed and amplified. The amount of cellular D8 mRNA is estimated by comparing the intensity of the resulting PCR products on an agarose gel. The method has been applied to quantify D8 and topoisomerase II mRNA concentrations in small cell lung cancer (SCLC) derived cell lines. The D8 gene is a candidate tumor suppressor gene, localized in the chromosomal region 3p21, the common deletion region in lung cancer. This study showed that in the majority of cell lines the amount of D8 transcripts is less than 3% as compared to normal lung tissue. In addition we investigate a possible correlation of topoisomerase II levels with resistance to cytotoxic drugs.
111
A PCR AIDED TRANSCRIPT TITRATION ASSAY TO QUANTIFY mRNA LEVELS IN CELLULAR RNA. Apke van den Bera (1), Klaas Kok (1), Egbert Smit (2), Charles H.C.M. Buys (1) Departments of Medical Genetics (1) and Pulmonology (2), University of Groningen, Groningen, The Netherlands. We developed a general method to quantify the amount of a specific mRNA in a given amount of total cellular RNA. The method involves the construction of a recombinant cDNA clone by ligating a small restriction fragment into a unique restriction site of the cellular cDNA clone. Two primers were selected on either site of this restriction site. Upon PCR analysis, the recombinant cDNA gives rise to a slightly longer PCR product than the cellular cDNA. Recombinant RNA, to be used as an internal standard, was synthesized in vitro, using the recombinant cDNA clone as a template. Samples containing a fixed amount of cellular RNA and varying amounts of recombinant RNA are reverse transcribed and amplified. The amount of cellular D8 mRNA is estimated by comparing the intensity of the resulting PCR products on an agarose gel. The method has been applied to quantify D8 and topoisomerase II mRNA concentrations in small cell lung cancer (SCLC) derived cell lines. The D8 gene is a candidate tumor suppressor gene, localized in the chromosomal region 3p21, the common deletion region in lung cancer. This study showed that in the majority of cell lines the amount of D8 transcripts is less than 3% as compared to normal lung tissue. In addition we investigate a possible correlation of topoisomerase II levels with resistance to cytotoxic drugs.