Quantification of Bacterial Transcripts during Infection Using Competitive Reverse Transcription-PCR (RT-PCR) and LightCycler RT-PCR

One-Step Reverse Transcription PCR (RT-PCR). Fatemeh Mehrpooyan*

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.

The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new en...

Reproducibility in the quantification of mRNA levels by RT-PCR-ELISA and RT competitive-PCR-ELISA.

The use of reverse transcription (RT) PCR for relative quantitation of gene transcripts relies on the reproducibility of the individual RT, PCR and product measurement steps. Semi-competitive RT-PCR (RT-cPCR) uses an internal competitor template in the PCR step to improve quantitation. We have surveyed the reproducibility of RT, PCR, RT-cPCR and measurement, amplifying the glyceraldehyde-3-phos...

Improved mRNA quantitation in LightCycler RT-PCR.

BACKGROUND Real-time polymerase chain reaction (PCR) utilizing the LightCycler and similar systems is an increasingly used technique for quantitative reverse transcription (RT)-PCR of mRNA levels from genes of immunologic interest. A commonly encountered limitation with these systems is that the fluorescence induced by SYBR Green (a fluorophore that binds double-stranded DNA) can result from pr...

Quantitative RT-PCR using a PCR-generated competitive internal standard.

One approach to quantitate RNA is the use of a known number of competitive internal standard molecules that are reverse-transcribed and subsequently co-amplified under the same reaction conditions as the target sequence (3). This technique, called competitive reverse transcription polymerase chain reaction (cRT-PCR) has been applied to monitor the level of hepatitis C virus (HCV) RNA in patient...