Author: Magda

24 Jun A special analytical design getting cousin quantification during the actual-go out RT–PCR

Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.

Introduction

Opposite transcription (RT) with this new polymerase chain impulse (PCR) 's the way of substitute for analyse mRNA term produced from various provide. Real-go out RT–PCR is highly painful and sensitive and lets quantification off rare transcripts and you may quick alterations in gene term. Also so it, you can do, comes with the needed precision and you will supplies legitimate and additionally fast quantification efficiency.