#QPCR DATA ANALYSIS PROGRAMS HOW TO#

6-ħ Wide Dynamic Detection Range DEFINITION: The range of initial template concentrations over which accurate measurement can be achieved HIGX1A Gene NTC At least 7 log difference range can be detected to accommodate varying initial sample input amounts/gene expression levels, in contrast to <2 log difference by traditional PCR (End point analysis by gel electrophoresis) -7-Ĩ Wide Range of Applications RNA DNA Gene Expression Profiling Analysis Microarray Validation mirna Expression Profiling Analysis Gene Regulation Genetic & Epigenetic SNP Genotyping & allelic discrimination Somatic Mutation Analysis Copy Number Detection/Variation Analysis Pathogen Detection Viral Quantification -8-ĩ Summary of The Advantages Detect the reporter signals at early & exponential phase, in real time! Precisely detect the amplicon during each cycle proportional to the initial template amounts. (A) same starting amounts of RNA (B) Different starting amounts. 3-Ĥ Understanding Reaction Kinetics in PCR Theoretically, the PCR reaction doubles the DNA amount after completing each cycle exponential reaction! The reaction, however, finally tails off and reaches a plateau -4-ĥ Understanding Kinetics in PCR Amplification Plot (Linear scale) Plateau End point data collection at plateau (gel analysis) Fluorescence Signal Reactions start varying due to reagent depletion & decreased PCR efficiencies (enzyme activity, More product competing for primer annealing Real time PCR does early phase detection at the exponential state Precisely proportional to input amounts -5-Ħ Real-Time qpcr vs End Point Detection (cont.) Look different Look similar The RNA was reversed transcribed to cdna followed by real time or end point analysis by gel electrophoresis. This detection occurs during the accumulation of the PCR product with each cycle of amplification, thus allows monitoring the PCR reaction during early & exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. It is based on detection and quantification of fluorescence emitted from a reporter molecule at real time.

#QPCR DATA ANALYSIS PROGRAMS HOW TO#

1 Introduction To Real Time Quantitative PCR (qpcr) SABiosciences, A QIAGEN CompanyĢ The Seminar Topics The advantages of qpcr versus conventional PCR Work flow & applications Factors influencing the performance of a qpcr assay Reporter chemistry Terms commonly used Ct method for relative quantifications How to set up a qpcr reaction Data output & analysis -2-ģ What Real-Time qpcr Stands for? Real time qpcr is the most sensitive and reliable method for detection and quantification of nucleic acids (DNA, cdna, & RNA) levels.

Qpcr data analysis programs