qPCR
qPCR is a powerful tool for measuring DNA/RNA. Explore the module below for qPCR planning and troubleshooting tips.
This interactive subway map outlines the steps in a typical qPCR workflow and links out to more detailed information and suggested reagents.
Click through to discover low-waste consumables and digital tools that will take your qPCR experiments to the next level.
Note: The information on this site applies to qPCR using intercalating dyes not fluorescent probes. The intercalating dye method is simpler and sufficient for most applications
From PCR...
- DNA is amplified through iterative rounds of denaturation, annealing, and extension.
- Performed in a thermal cycler that cycles between the different temperatures required for each step.
...to qPCR
- Fluorescent dyes or oligos are used to quantify each molecule of DNA that is amplified.
- Performed in a special type of thermal cycler that measures fluorescence
1. Extraction
Whether you want to use qPCR to quantify RNA or DNA, you’ll need to first extract your target from your starting culture/tissue. There are a variety of kits and protocols that can be used for DNA extraction with varying costs and complexity. Fundamentally, they all facilitate three steps:
- Cell lysis and protein degradation
- DNA/RNA precipitation
- DNA/RNA purification
The leading edge of extraction technologies combine these three steps into one for faster, cleaner, simpler extractions.
- Macherey-Nagel NucleoSpin Virus Kits
- MicroGEM PDQex Nucleic Acid Extractor
2. qPCR Prep
Most qPCR reactions are composed of the same basic ingredients:
- The DNA or cDNA template
- primers that will anneal to the template to begin extension
- primers that will anneal to the template to begin extension
- DNA polymerase
- DNA polymerase
- Perkin Elmer Chemagic 360
- Macherey-Nagel NucleoMag Pathogen Kits
3. Reverse transcription
If your starting template is RNA, the RNA will need to be reverse transcribed into cDNA. This can be accomplished in a separate reverse transcription reaction. Pioneering reagent kits enable reverse transcription and qPCR in a single tube.
- PCRBIO SyGreen 1-Step Detect Lo-ROX
- UltraScript 2.0 cDNA Synthesis Kit Separate Oligos
4. Amplification
Like regular PCR reactions, the qPCR reaction will proceed in three steps:
- Denaturing: The double stranded DNA is heated until the two strands separate
- Annealing: primers/oligos anneal to the denatured DNA
- Extension: polymerase uses free nucleotides to reconstruct the second strand
In qPCR reactions, fluorescent dyes bind to the molecules of DNA as they’re amplified. Real-Time thermal cycles cycle through the temperature changes needed to transition through the three PCR steps while simultaneously measuring fluorescence.
- PCRBIO HiFi Polymerase
5. Analysis
Relative DNA/RNA levels measured by the thermal cycler are reported in graphs plotting fluorescence versus cycle number.
By creating a standard curve for a series of samples with known concentrations, the relative value can be converted into an absolute concentration.
The efficiency and cycle threshold (CT) values will help you determine the quality of the reaction. CT represents the threshold point at which the presence of the amplified molecule is detectable above background. For more information, see technical notes.
Featured analysis products
- Douglas Scientific IntelliQube Automated qPCR System