Quantitative real-time PCR (qPCR) is a versatile technology, and widely used for applications such as clinical diagnostics, gene transcription analysis and agricultural breeding programs.
A well-designed assay can be highly sensitive and specific, but ensuring the integrity of your results involves careful planning and optimization, from sample preparation and design of the reaction mix and controls, through to appropriate data analysis.
Download this guide to explore tips on:
- Sample preparation
- Assay design
- Quality control
- Data analysis
How To Guide
Perfect Your qPCR
Katie Minns, PhD
Quantitative real-time PCR (qPCR) is a versatile technology, and widely used for applications such as
clinical diagnostics, gene transcription analysis and agricultural breeding programs.
A well-designed assay can be highly sensitive and specific, but ensuring the integrity of your results
involves careful planning and optimization, from sample preparation and design of the reaction mix
and controls, through to appropriate data analysis.
So, whether you’re just getting started with qPCR or understand the basics, here are some tips to
boost the reliability and consistency of your results.
Sample preparation
Getting started with a suitable sample
One of the first things to consider when planning a qPCR experiment is choosing your sample.
Understand any relevant cellular mechanisms and be sure that the nucleic acid sequence you are
targeting will be present in the sample chosen and really answers the question you’re investigating.
Clinical specimens such as blood and sputum can be difficult to work with due to inhibitors1 and vis
cosity,
while plant samples require mechanical grinding steps. To isolate the nucleic acids from your
chosen matrix, you may require a specialized kit or additional processing steps.
If you’re working with RNA, be aware that this degrades quickly.2 Use fresh samples where possible,
.2
keep them cold and quickly neutralize the ribonuclease enzymes that degrade RNA.
Purify your nucleic acids
The quality of your results will be impacted by the quality of the nucleic acids that you start with in
your sample, so purification steps are important. Spin columns offer a convenient and rapid purifi
cation
option. Magnetic bead-based technologies offer a more gentle method with no centrifugation,
minimizing shearing of long-stranded nucleic acids
.3
Use a spectrophotometer to check the quality and concentration of your starting material. The
amount of nucleic acid you’ll need to add to your reaction mix will depend on the complexity/size of
your target. Plasmid DNA could require as little as 0.01–1 ng, while genomic DNA (gDNA) may need
up to 0.1–1 µg as there will likely be fewer copies of your target in a given quantity.4 Similarly for
4
RNA, you’ll need higher concentrations for total RNA, and lower concentrations for messenger RNA.
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