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

Gel Purification of PCR Products. Fatemeh Mehrpooyan*

Use of elevated reverse transcription reaction temperatures in RT-PCR.

The use of reverse transcriptase coupled with the polymerase chain reaction (RT-PCR) has been proven to be a valuable tool in the synthesis and isolation of cDNA clones as well as the quantification of rare messenger RNA species. While a number of different approaches can be used to permit gene-specific cDNA synthesis and amplification, a recurring problem is one of high background signal (i.e....

One-Step RT-PCR protocols improve the rate of dengue diagnosis compared to Two-Step RT-PCR approaches.

Dengue is the most important arboviral disease transmitted to humans. In our laboratory, we have been working on the standardization of the polymerase chain reaction (PCR) diagnosis of this disease. In this work, we compared five commercial kits regularly used on reverse-transcription polymerase chain reaction (RT-PCR) protocols: two Two-Step kits (SuperScript II RT/Super Mix kit and reverse tr...

One-step RT-PCR for screening microdissected tissue samples.

The technology of laser-capture microdissection (LCM) enables the easy extraction of single cells or defined groups of cells from a tissue section (1,2). This technique uses a laser pulse to activate a plastic polymer, which binds to and captures cells from a frozen or paraffin-embedded tissue section. Captured cells present on this solid support can then be used for nucleic acid and/or protein...

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...

One-step triplex PCR/RT-PCR to detect canine distemper virus, canine parvovirus, and canine kobuvirus.

To rapidly distinguish Canine distemper virus (CDV), canine parvovirus (CPV), and canine kobuvirus (CaKoV) in practice, a one-step multiplex PCR/RT-PCR assay was developed, with detection limits of 102.1 TCID50 for CDV, 101.9 TCID50 for CPV and 103 copies for CaKoV. This method did not amplify nonspecific DNA or RNA from other canine viruses. Therefore, the assay provides a sensitive tool for t...