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Thursday, January 3, 2013

Real Time PCR (qpCR) Quantitation - Methods and Types of analysis - Absolute vs Relative Quantitation



A typical real time PCR graph - qPCR graph showing Ct (cycle threshold) or Cq (quantification cycle) Vs Flourescence


Real-Time PCR (qPCR) experiment results can be analyzed using these methods,

Methods of Analysis
  • Absolute quantitation
  • Relative quantitation
Absolute quantitation:

The absolute quantitation assay is used to quantitate unknown samples by interpolating their quantity from a standard curve.

Absolute quantitation is a method of qPCR analysis in which it gives the measure the copy number in the sample. Absolute quantitation is useful in determining the viral load and also it helps in comparison of results among different labs.

For absolute quantitation, absolute quantities of the standards must first be known by some independent means.

Currently absolute quantitation not feasible because there is no reliable methods for preparing, quantitating and storing standards, especially RNA standards. There are no traceable NIST standards (NIST: National Institute of Standards and Technology).

The calculation methods used for absolute quantification are:
  • Digital PCR Method
  • Standard Curve Method
since standards are not available other best option is to synthesize template and make it to a known Ct value and such method is called "semi quantitative PCR".Oligo's can be synthesized separately and can be assembled by assembly PCR.

Relative Quantitation:

In relative quantitation method changes in gene expression of a given sample relative to another reference sample is analyzed.

Relative quantification can be performed with data from all real-time PCR instruments. The calculation methods used for relative quantification are:
  • Standard Curve Method
  • Comparative Ct Method
Relative quantification method can be used to determine fold increases and decreases in gene expression and there is no need to over optimize the efficiencies.

In relative quantification any sample that can be used as a comparison for other samples-"calibrator", and a serial dilution of the calibrator to give a standard curve in terms of 1x, 2x, 10x, etc

Comparative Ct method is based on mathematical determination of relative quantities, No standard curve is needed and this method has higher throughput and is Best used when particular ratios are expected or are verifying a trend.

For comparative quantitation, calibrator sample used as a 1x standard and the samples that are prepared identically. Ideally, if normalizing the results, your Gene of Interest (GOI) and the normalizer will have the same efficiency.

Comparative Ct Quantitation Calculation

Ct GOI - Ct norm = ∆Ct

∆Ct Sample - ∆Ct Calibrator = ∆∆Ct

Relative quantity = 2-∆∆Ct

Definitions

Standard: A sample of known concentration used to construct a standard curve.
Calibrator: A sample used as the basis for comparative results.

References
Methods for qPCR Analysis, Renée Horner, Ambion, Inc.

Absolute vs. Relative Quantification for qPCR, Invitrogen Technical Resources

REAL TIME PCR, Dr Margaret Hunt, Microbiology and Immunology online, university of south carolina school of medicine.

Essentials of Real time PCR, Applied Biosystems