Vol. 35, No. 6 (2003) BioTechniques 1141 the ABI PRISM® 7000 and the TaqMan Universal PCR Master Mix protocols (Applied Biosystems). After discovering that the RNA from the thecal cells had vascular endothelial growth factor (VEGF) amplification and that RNA from granulosa cells had no VEGF amplification, the 18S rRNA amplification (TaqMan Pre-Developed Assay Reagents; Applied Biosystems), whic...

3.Higuchi, R., B. Krummel and R.K. Saiki. 1988. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16:7351-7367. 4.Sarkar, G. and S.S. Sommer. 1990. The “megaprimer” method of site-directed mutagenesis. BioTechniques 8:404-407. 5.Smith, M. 1985. In vitro mutagenesis. Annu. Rev. Genet. 19:423-462. 6.Upende...

With the introduction of real-time, fluorescence-based 5′ nuclease PCR (3,4) and instruments such as the ABI PRISM 7700 (TaqMan) sequence detector (Applied Biosystems, Foster City, CA, USA), quantitative RT-PCR is now a widely accepted method for measuring gene expression levels. Quantitative RTPCR is a sensitive technique and is particularly useful for the analysis of samples containing limi...

A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6...