無標題文件

Activity

Date

Time

Place

Workshop-A

9/1( 六 )

9:00~12:00

National Yang-Ming University , Taipei

Workshop-B

9/3( 一 )

9:00~16:30

303 classrooms, Department of Chemical Engineering , National Chung Cheng University, Chia-yi

Symposium-Day1

9/4( 二 )

10:10~18:00

禮堂國際會議廳 , National Chung Cheng University, Chia-yi

Symposium-Day1

9/5( 三)

9:00~12:30

禮堂國際會議廳 , National Chung Cheng University, Chia-yi

Abstract

In this workshop I'll discuss the polymerase chain reaction (PCR), at first generally, and then in quantitative terms. Initially, quantitation in the context of PCR simply meant quantifying the amount of end product of the reaction, and comparing amounts, but about 10 years ago, a novel approach was developed, now called quantitative real-time PCR, here abbreviated QRT-PCR.

Where standard PCR quantifies a product at end-point after exponential amplification, QRT-PCR continuosly monitors an accumulation of product in real-time. Measurements are made in the exponential phase of the PCR reaction and avoid the effect of limiting reagents, amplicon re-annealing, post-PCR processing and cycling variability of end-point analysis.

This assay is mainly used for accurate quantitation of mRNA, and so is preceded by a reverse-transcription step, so RT-PCR sometimes means "reverse-transcription" PCR, not "real-time" PCR. Take care when reading!

Reverse transcription followed by the polymerase chain reaction is the method of choice for quantifying rare transcripts in biological samples. A key assumption underlying the absolute quantification of transcripts is similar amplification efficiencies of all external standards and samples. However, efficiencies can vary between individual reactions, a problem that can be magnified when quantifying transcripts of low abundance.

All QRT-PCR calculations make use of the efficiency of the polymerase chain reactions in one way or another. I'll describe some of the common approaches, including the so-called delta-delta Ct method. Many methods simply assume the efficiency remains at 1, i.e. that a doubling takes place at each cycle until the cycle threshold is reached; others make use the slope of a fitted standard curve, while yet others estimate and use the efficiencies of individual reactions in various ways.

I will review the literature on efficiencies, pointing out the difficulties in obtaining a single efficiency figure for an individual reaction. The reason is simple and widely accepted: there is clear evidence from the cumulative intensity values that the replication efficiency changes as the reaction proceeds, raising doubts about the value of speaking about "the efficiency" of a single reaction. Further, it is extremely hard to give stable estimates of efficiency during the reaction. Finally, I will discuss how efficiency considerations might be able to help in carrying out the primary task of qrtPCR, which is to provide estimates of absolute or relative concentrations of DNA molecules.

If time permits, I will conclude my workshop with a brief discussion of methods of detecting alternative splicing using data from the Affymetrix Human Exon 1.0 ST Array.