How to optimize qPCR using SYBR Green Assays--Taq Talk Episode 21

Learn about RT bias sources including reverse transcriptase efficiency, RNA integrity, and secondary structure that affect cDNA generation. Test for RT bias by reverse transcribing two-fold RNA dilutions and running qPCR standard curves to verify linearity with target slope of -3.323.

Use software like Applied Biosystems Primer Express to design primers that are 20 nucleotides long with 30-70% GC content. Ensure the last five 3' nucleotides contain no more than two G or C bases, and design amplicons between 50-150 base pairs.

Run multiple qPCR reactions testing three different concentrations of forward and reverse primers in a matrix format. For PowerTrack SYBR Green master mix, test primer concentrations in the 300-800 nanomolar range.

Analyze the Ct values and run melting curve (disassociation curve) analysis for each primer concentration combination. If primer dimers are detected, either redesign the primers or alter cycling temperatures to eliminate them.

Run a standard curve with at least five logs of input DNA and use instrument software to calculate PCR efficiency. Confirm efficiency is within the 90-110% range for acceptable assay performance.

Use Applied Biosystems TaqMan master mixes and pre-designed assays as an alternative if primer design and validation seem too complicated. This removes primer design variables and ensures optimal primer sets.