Two-step qPCR.

Materials

• SuperScript III (Thermo Fisher Scientific 18080051)
• PowerUp SYBR Green MasterMix (Fisher Scientific A25741)
• qPCR sub-skirted plate (MIDSCI AVT3890)
• Thermal sealing film (Excel Scientific TS-RT2RR-100)

Preparation

Two-step qPCR starts with RNA that has been DNase-treated and quantified with Qubit. Follow the RNA extraction protocol for C. elegans or for parasites.

Use the Invitrogen SuperScript III kit to perform first-strand cDNA synthesis:

• Do not freeze RNA between extraction and first-strand synthesis — perform synthesis immediately after quantification.
• Include a non-experimental RNA sample from the same species and stage as your experimental samples. Stock RNA samples from the FR3 work well.
• If performing relative transcript quantification, normalize the RNA input based on the Qubit quantification.
• Use a 1:1 mixture of oligo(dT) and random hexamer primers, and create primer and enzyme master mixes to use across all samples.
• Perform the optional RNase H digestion to remove remaining single-stranded RNAs.
• Freeze at -20°C until ready for the qPCR.

Before optimizing your primers, validate that they work and produce the expected amplicon length by performing normal PCR on a plasmid and/or cDNA template.

Book the qPCR machine on the morning of the reaction.

Primer optimization

qPCR primers must be optimized for each new RNA/cDNA template. Optimization involves primer dilutions to find the optimal primer concentration for each primer set and template combination. Use the same stage, non-experimental cDNA sample at the same concentration as your experimental samples — best practice is to include this sample in your cDNA synthesis reaction. If you are working with C. elegans, RNA is not as precious; with parasites it is paramount not to waste RNA from experimental samples. The PowerUp SYBR Green Master Mix kit recommends trying 300, 500, and 800 nM final concentrations, but you can add additional lower concentrations as well.

Reference genes