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Relative And Absolute Quantitative Real Time Pcr

2201 words - 9 pages

Relative and Absolute quantitative Real-Time PCR (qRT-PCR)

Introduction

Quantitative Real-Time in PCR (qRT-PCR)

The polymerase chain reaction (PCR) is a revolutionized technology used in molecular biology for detection and amplification of DNA generating thousands to millions of copies of a particular DNA sequence. Quantitative real-time reverse transcription-polymerase chain reaction PCR (RT-QPCR) is regarded as the golden standard technique in molecular biology and has been seen as a bench-marking analytic for DNA and mRNA detection, this technique is also used in a wide variety of bio-analytical science areas (Burns et al, 2005). RT-QPCR is a technique that simultaneously monitors, amplifies and quantifies nucleic acid accumulation in real time and is characterised as a high quality performance technique associated with high throughput, reproducibility, specificity and sensitivity. There are a number of steps involved in qRT-PCR which include RNA extraction, cDNA synthesis, PCR amplification and normalization with suitable internal standard or reference gene. Different selection methods, internal standards all have an effect on the reliability of qRT-PCR (Zhang et al, 2012). RT-qPCR validates and analysis data in basic PCR fluorescent thermal cycle software which depends on either absolute or relative quantification of PCR data, this validation system requires the comparison of sample to standard DNA or to reference genes, respectively (Roussel et al, 2007).

Relative quantification Real-Time PCR (qRT-PCR)

Relative quantification has been widely studied in cytokine or enzymes to evaluate gene expressions changes; this type of quantification omits the use of standards and allows the comparison of two experiments which is an advantage of using this type of quantification as different experimental comparison can be exploited. In relative quantification a comparative Ct or standard curve method is used to measure the reference gene expression normally in relative time using a calibrator. Quantification using this type of method may miss relative biological information compared to absolute method which relies on standard measurements or copy numbers of a particular target and therefore is considered to be more informative and reliable in comparisons (Dhanasekaran et al, 2010). Relative quantification as already discussed allows the comparison of the target gene to reference gene within the same sample, in this method the reference gene is normally consistently expressed across the experimental sample e.g. developmental series, tissue set and experimental regime (Sellars et al, 2007). This type of quantification as opposed to absolute provides a direct answer to changes or differences in gene expression as well as uses multiple gene comparison. The most used housekeeping genes in relative qRT-PCR experiments include 18S rRNA, β-actin, elongation factorα and GAPDH, however recent studies have shown variability in expression levels of...

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