Your controls usually say what went wrong. A positive control, a no-template control, a no-RT control and one dilution of the sample separate template, inhibition and primer problems.
Range: qPCR and RT-PCR reagents3 minute readUpdated
| Result | Meaning | Next step |
|---|---|---|
| Positive control fails | Mix, primers, cycling or instrument | Check dye channel, ROX setting, cycling program |
| Positive control works, samples do not | Template or reverse transcription | RNA quality, RT step, input amount |
| Signal in the no-template control | Contamination or primer dimers | Inspect the melt curve |
| Signal in the no-RT control | Genomic DNA amplifying | DNase treat; span an exon junction |
| Replicates scatter, Cq above about 35 | Too few template copies | Add more input; expect noise at low copy |
A later Cq than expected means less amplifiable template, or an assay that is not doubling each cycle. Efficiency is calculated from a dilution series: a slope of about -3.3 is 100%, and 90–110% (slopes of about -3.6 to -3.1) is the usual acceptable window.
With a dye such as SYBR Green, a clean assay shows one sharp melt peak. Primer dimers appear as a separate peak at a lower temperature, usually in the no-template control too. A peak at the same temperature as your product in the no-template control means real contamination.
When only one component is in doubt, replace it rather than the whole workflow. The qPCR and RT-PCR range covers premixes, Taq and reverse transcription kits. See PCR vs qPCR vs RT-qPCR for which reagent each method needs.
Check the positive control first. If it also fails, look at the dye channel, the ROX setting, the cycling program and the master mix. If it works, the problem is the template or the reverse transcription.
Either contamination or primer dimers. In a dye-based assay, a melt peak at the same temperature as the product means contamination; a lower-temperature peak means primer dimers.
Usually 90–110%, equal to a standard-curve slope of about -3.6 to -3.1. A slope of about -3.3 corresponds to 100% efficiency.
Run the sample neat and diluted. Each 10-fold dilution should delay Cq by about 3.3 cycles. If the diluted sample comes up as early as or earlier than the neat one, inhibitors are likely.
For research use only. Protocols here are general outlines: follow your kit manual and your institution’s safety rules. See our editorial policy.
Products for this workflow
Taq DNA polymerase, PCR and qPCR premixes, and reverse transcription kits for cDNA synthesis.
qPCR / RT-PCR
A 2X qPCR master mix looks like a commodity until it does not match your instrument or chemistry. This guide covers dye vs probe, ROX levels, hot-start, setup volumes and how to validate efficiency.
4 minute read
qPCR / RT-PCR
End-point PCR tells you whether a sequence is there, qPCR tells you how much, and RT-qPCR does the same for RNA. Here is what changes between them and the reagent list for each.
4 minute read
DNA and RNA extraction
A poor A260/230 usually means salt or phenol carry-over rather than bad RNA. Low yield usually starts at lysis. Here is how to read the numbers and what to change.
3 minute read
Send the product names or catalogue numbers, quantities and delivery country. We'll confirm availability, lead time and the documents for qpcr and rt-pcr reagents.