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- Lintula, S, et al.
(författare)
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Relative concentrations of hK2/PSA mRNA in benign and malignant prostatic tissue
- 2005
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Ingår i: The Prostate. - : Wiley. - 0270-4137 .- 1097-0045. ; 63:4, s. 324-329
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND. Prostate-specific antigen (PSA/KLK3) and human kallikrein 2 (hK2/KLK2) belong to the human kallikrein gene family. These two highly homologous genes are specifically expressed in the prostate under androgen control. Expression of these is regulated by similar mechanisms but changes in their relative expression have been observed in prostate cancer. METHODS. We determined the relative levels of PSA and hK2 mRNA in benign and malignant prostate tissue using a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. The mRNA of PSA and hK2 are reverse transcribed and amplified in one reaction with the same primers. RESULTS. The variation in the ratio of hK2/PSA mRNA was remarkably small, the difference between the highest and lowest values being three-fold. The ratio was significantly higher in WHO grade 2 compared to normal or benign prostatic hyperplasia tissue (P=0.032 and P=0.035, respectively) and in grade 3 compared to normal or benign prostatic hyperplasia tissue (P=0.006 in both). CONCLUSIONS. The new quantitative RT-PCR technique facilitates very accurate quantitation of the relative mRNA levels of homologous genes. Using this method we have shown that the ratio of hK2/PSA mRNA is higher in cancerous than in benign prostatic tissue.
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- Feng, L, et al.
(författare)
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Technique for strand-specific gene-expression analysis and monitoring of primer-independent cDNA synthesis in reverse transcription
- 2012
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Ingår i: BioTechniques. - : Future Science Ltd. - 1940-9818 .- 0736-6205. ; 52:4, s. 263-
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Tidskriftsartikel (refereegranskat)abstract
- Primer-independent cDNA synthesis during reverse transcription hinders quantitative analysis of bidirectional mRNA synthesis in eukaryotes as well as in cells infected with RNA viruses. We report a simple RT-PCR-based assay for strand-specific gene-expression analysis. By modifying the cDNA sequence during reverse transcription, the opposite strands of target sequences can be simultaneously detected by postamplification melting curve analysis and primer-initiated transcripts are readily distinguished from nonspecifically primed cDNA. We have utilized this technique to optimize the specificity of reverse transcription on a panel of 15 target genes. Primer-independent reverse transcription occurred for all target sequences when reverse transcription was performed at 42°C and accounted for 11%–57% of the final PCR amplification products. By raising the reaction temperature to 55°C, the specificity of reverse transcription could be increased without significant loss of sensitivity. We have also demonstrated the utility of this technique for analysis of (+) and (-) RNA synthesis of influenza A virus in infected cells. Thus, this technique represents a powerful tool for analysis of bidirectional RNA synthesis.
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