Wikipedia:United States Education Program/Courses/JHU MolBio Ogg 2012/Section 81/Group 81C/WIP

Introduction

Y Drafted & moved to the actual article.

History

Y Drafted & moved to the actual article.

Principles

Y Drafted & moved to the actual article.

Application

Under draft

Old contents to be integrated - lacking references so we need to validate or replace

The exponential amplification via reverse transcription polymerase chain reaction provides for a highly sensitive technique in which a very low copy number of RNA molecules can be detected. RT-PCR is widely used in the diagnosis of genetic diseases and, semiquantitatively, in the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression. Northern blot analysis is used to study the RNA's gene expression further. RT-PCR can also be very useful in the insertion of eukaryotic genes into prokaryotes. Because most eukaryotic genes contain introns which are present in the genome but not in the mature mRNA, the cDNA generated from a RT-PCR reaction is the exact (without regard to the error prone nature of reverse transcriptases) DNA sequence which would be directly translated into protein after transcription. When these genes are expressed in prokaryotic cells for the sake of protein production or purification, the RNA produced directly from transcription need not undergo splicing as the transcript contains only exons. (Prokaryotes, such as E. coli, lack the mRNA splicing mechanism of eukaryotes).

RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA, such as Influenzavirus A and retroviruses like HIV.

Challenges

To be drafted during Unit 13

Protocol

To be drafted during Unit 14

Publication Guidelines

Quantitative RT-PCR assay is considered to be the gold standard for measuring the number of copies of specific cDNA targets in a sample but it is poorly standardized. (1) As a result, while there are numerous publications utilizing the technique, many provide inadequest experiental detail and use unsuitable data analysis to draw inappropriate conclusions. Due to the inherent variability in the quality of any quantitave PCR data, reviewers not only have a difficult time evaluating these manuscripts, the studies also become impossible to replicate. (2) Recognizing the need for the stardardization of the reporting of experimental conditions, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE, promounced mykee) quidelines have been published by the international consortium of academic scientists. The MIQE guidelines describe the minimum information necessary for evaluating qPCR experiments that should be required for publication for encouraging better experimental practice and ensuring the relevance, accuracy, correct interpretation, and repeatability of quantitative PCR data. (3)

Besides reporting guidelines, the MIQE stresses the need to standardize the nomenclature associated with quantitative PCR to avoid confusion; for example, the abbreviation qPCR should be used for quantitative real-time PCR and RT-qPCR should be used for reverse transcription-qPCR, and genes used for normalisation should be referred to as reference genes instead of housekeeping genes. It is also proposes that commercially derived terms like TaqMan® probes should not be used but instead referred to as hydrolysis probes. Additionally, it is proposed that quantification cycle (Cq) be used to describe the PCR cycle used for quantification instead of threshold cycle (Ct), crossing point (Cp), and takeoff point (TOP), which refer to the same value but were coined by different manufacturers of realtime instruments. (1)

The following checklist outlines the MIQE guidelines that must be submitted with the manuscript and should be published either in abbreviated form or as an online supplement (3):

Copy table from http://www.rdml.org/miqe.php

References:

(1)

(2)

(3)