This workshop is designed for both beginners and those who are already working with the technique but wishing to learn appropriate statistics that can be applied to the RT-PCR data. It is a comprehensive workshop that covers how the technology works, different types of detection technologies, designing primes and probes, optimizing new assays with stable reference genes and proper normalization. The focus will be on hands-on lab work for relative quantification using SYBR green intercalating-dye chemistry and standard curve and melt curve to validate RT-PCR assay performance. After the workshop, participants will be able to plan and perform RT-PCR experiments themselves, as well as interpret and analyze data.

Lecture 1:   Fundamentals and applications of Real-Time PCR (qPCR)

Lecture 2: Sample preparation, assessment and reverse transcription prior to qPCR compliance with MIQE guideline

Practical 1: RNA extraction

Practical 2: RNA quantification and integrity checking using automated electrophoresis system

Analysis of Experion results

Practical 3: Reverse transcription – cDNA synthesis
Lecture 3:   Assay design guideline

Demo:       Primer/probe design with Beacon Designer Software (Sybr green assay & TaqMan Probe Assay)

Lecture 4:   qPCR assay optimization and validation compliance with MIQE guideline

Lecture 5:   Gene expression (reference gene, M value, useful publication)

Practical 4: qPCR assay setup (Std Curve & Gene Expression)

Lecture 6:   Application specific talks – gene silencing (invited speaker)

Lecture 7:   Application of micrarray data with qPCR (invited speaker)

Lecture 8:   Application specific talks – absolute quantification in viral load

Practical 5: qPCR software setup and data analysis – Troubleshooting

Lecture 8:   SPSS software to look for significant value

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