Preparing polyA-Containing RNA Internal Standards for Multiplex Competitive RT-PCR


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Preparing polyA-containing RNA internal standards for multiplex competitive RT-PCR.

PCR-based strategies such as competitive RT-PCR or semiquantitative RTPCR are commonly used for quantitating low-abundance mRNA transcripts (4,7–9). Competitive RT-PCR relies on amplifying the same target with a known amount of a competitor/reference sequence and comparing the relative amounts of the two PCR products. To improve the sensitivity and accuracy of these methods, often expensive and...

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Serial competitive RT-PCR using multiple standards.

PCR is widely used for the amplification of DNA and reverse-transcribed RNA. In recent years, real-time PCR has been introduced as a new technique for mRNA quantitation. However, because of the high cost of real-time PCR equipment and supplies, especially using custom-made probes, quantitative RT-PCR is a valuable alternative method. Several quantitative PCR protocols describe the use of a sing...

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Competitive RT-PCR: Preparation of Competitor RNA.

INTRODUCTION The first step in competitive RT-PCR is the synthesis and purification of the synthetic competitor. This is an RNA molecule designed to be reverse-transcribed and PCR-amplified with the same efficiency as the endogenous transcript of interest. Once the competitor molecule has been prepared, as described in this protocol, competitive PCR can be carried out, as described in Comptetit...

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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...

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عنوان ژورنال: BioTechniques

سال: 2000

ISSN: 0736-6205,1940-9818

DOI: 10.2144/00293bm09