Real time PCR and it's functions in diagnosis

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Log fluorescence (Rn)

Slide 11

Using the PCR Equation

Xn = X0(1 + E)n

Xn =PCR product after cycle n

X0 =initial copy number

E =amplification efficiency

n =cycle number

Xn

X0

cycle number

A

Introduction

Advantage disadvantages

Product Detection

in medicine

Conclusion

Reference

Real Time PCR

A difference of 0.1 in amplification efficiency create a five-fold difference in the final ratio of PCR products after 30 cycles.

Xn = X0(1+E)n

Case 1: E = 0.9 Case 2: E = 0.8

Xn = 100 (1+0.9)30

Xn = 100 (1+0.8)30

Xn = 2.3 x 1010

Xn = 4.6 x 109

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Slide 12

Introduction

Advantage disadvantages

Real Time PCR

Product Detection

in medicine

Conclusion

Reference

Instruments, Accessories and Software

1 ) Light Cycler® Relative Quantification Software

The first commercially available software was the Light Cycler® Relative Quantification Software (2001).

2 ) REST

In 2002, the relative expression software tool (REST ) was established as a new tool.

3 ) Q-Gene

Recently a second software tool, Q-Gene, was developed, which is able to perform a statistical test of the real-time data.Q-Gene manages and expedites the planning, performance and evaluation of quantitative real-time PCR experiments.

4 ) qBASE

QBASE is an Excel®-based tool for the management and automatic analysis of real-time quantitative PCR data.

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Slide 13

5 ) SoFAR

The algoriths implemented in SoFAR (distributed by Metralabs) allow fully automatic analysis of real-time PCR data obtained with a Roch LightCycler® (Roche Diagnostics) instrument. The software yields results with considerably increased precision and accuracy of real-time quantification.

6 ) DART-PCR

DART-PCR (Data Analysis for Real-Time PCR) provides a simple means of analyzing real-time PCR data from raw fluorescence data.This allows an automatic calculation of amplification kinetics, as well as performing the subsequent calculations for the relative quantification and calculation of assay variability.

more…

Introduction

Advantage disadvantages

Product Detection

in medicine

Conclusion

Reference

Real Time PCR

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Slide 14

Real time PCR in comparison with other technical methods

Less time to getting results

Introduction

Advantage disadvantages

Product Detection

in medicine

Conclusion

Reference