qPCR and RT-qPCR

Quantitative PCR (qPCR) and reverse-transcription quantitative PCR (RT-qPCR) are rapid and sensitive techniques for detection of nucleic acids. qPCR is used to detect DNA and RT-qPCR requires an additional reverse transcription step to produce cDNA strands from an RNA-containing sample. The amount of DNA can then be determined quantitatively from the emission intensity of either a fluorescent probe or a fluorescent DNA binding dye. qPCR and RT-qPCR are "real-time" PCR techniques, that is they both involve the collection of data as the PCR process occurs and they combine amplification and detection into a single step.

Products
Background

Kits

Cat. No. Product Name / Activity
7478 Lyophilized OneStep RT-qPCR Master Mix
Lyophilized OneStep RT-qPCR Master Mix
7477 Lyophilized qPCR Master Mix
Lyophilized qPCR Master Mix

Other

Cat. No. Product Name / Activity
7246 Nucleic Acid Dye Green I
High affinity double-stranded DNA (dsDNA) probe; used in RT-LAMP and qPCR
What is qPCR?

Quantitative PCR (qPCR) and reverse-transcription quantitative PCR (RT-qPCR) are rapid and sensitive techniques for detection of nucleic acids. qPCR is used to detect DNA and RT-qPCR requires an additional reverse transcription step to produce cDNA strands from an RNA-containing sample. The amount of DNA can then be determined quantitatively from the emission intensity of either a fluorescent probe or a fluorescent DNA binding dye. qPCR and RT-qPCR are "real-time" PCR techniques, that is they both involve the collection of data as the PCR process occurs and they combine amplification and detection into a single step.

What are qPCR and RT-qPCR used for?

qPCR and RT-qPCR are used for the detection of DNA and RNA. They have a variety of applications including:

  • infectious disease research (identification and study of pathogens)
  • environmental analysis (water safety and testing)
  • food testing (safety, contamination, microbes, allergens)
  • gene expression studies
  • diagnostics
  • genotyping

What is the difference between PCR and qPCR?

PCR and qPCR are methods with a range of different applications in life science research. The most distinguishable difference is that the amplification of genetic material by qPCR can be followed in "real time" quantifying the amount of DNA in the reaction. Conversely, DNA amplified by standard PCR may only be visualized at the end of the reaction (Figure 1). Standard PCR has a variety of applications in molecular biology including cloning and gene mutagenesis experiments.


qPCR and RT-qPCR

The Difference Between qPCR and RT-qPCR

Figure 1: qPCR and RT-qPCR Schematic showing the difference between quantitative PCR and reverse-transcription quantitative PCR processes


Detection of RNA - one-step or two-step?

When amplifying an RNA target a reverse transcription (RT) step is required to generate complementary DNA (cDNA). This step is performed by a reverse transcriptase enzyme and a choice of primers including gene-specific primers, random oligomers and oligo-dT primers. Primers are selected based on whether the subsequent amplification of cDNA is performed in a one-step or two-step process. In one-step RT-qPCR, reverse transcription and PCR are performed in the same tube using gene specific primers. In two-step RT-qPCR, reverse transcription and PCR are performed separately with random oligomers and oligo-dT primers used to generate cDNA. In a two-step process multiple PCRs from a single RT reaction may be performed however one-step RT-qPCR offers easy handling, high reproducibility and reduced risk of contamination. While the goals of the experiment should dictate which method is used, one-step RT-qPCR is the most advantageous for researchers who are amplifying well-characterized targets with well-defined primers and probe sequences.

How is genetic material detected in qPCR and RT-qPCR?

There are several options for the quantitative measurement of replicated DNA. One option is to use a non-specific fluorescent dye that binds to the double-stranded DNA (e.g. Nucleic Acid Dye Green I Cat. No. 7246) and produces a signal proportional to the amount of DNA present. Another detection option is to use a hydrolysis probe and primers specific for a target DNA sequence. A hydrolysis probe (Figure 2) consists of a fluorophore and a quencher attached to an oligonucleotide that binds to a specific DNA sequence. During the qPCR process, the probe's nucleotide sequence anneals to the target DNA, and is then hydrolyzed by DNA polymerase to separate the fluorophore from the quencher, allowing the probe to fluoresce. The level of fluorescence is proportional to the amount of probe target sequence in the sample and can be used to calculate the number of copies of the DNA or RNA that were in the original sample.


Hydrolysis Probes

Detecting Nucleic Acids Using a Hydrolysis Probe

Figure 2: Hydrolysis Probe Schematic showing annealing of the probe and hydrolysis of the fluorophore and quencher, allowing the probe to fluoresce.


Lyophilized Master Mixes

The lyophilized master mixes are optimized, complete systems for quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) techniques using hydrolysis probes. The RT-qPCR master mix is a one-step system that offers a quick setup, easy handling, reduced pipetting and a reduced chance of sample contamination over two-step RT-PCR methods. The resuspended master mixes require only the addition of your cDNA/RNA, primers and probe to be qPCR or RT-qPCR ready.

The master mixes contain a thermo-stable TAQ Polymerase (and MMLV for the RT-qPCR master mix) as well as buffer, dNTPs, MgCl2 and stabilizers at concentrations optimized for the enzymes.

The qPCR and RT-qPCR master mix kit contents are optimized for the enzymes and lyophilized to stabilize the active components. This ensures that the master mixes can be stored and transported at room temperature (no cold shipping required) and are stable for 12 months at ambient temperatures, prior to resuspension. There is no significant effect on performance or loss of activity of the master mixes after repeated freeze-thawing (once resuspended the master mixes should be stored at -20°C for later use). The stable formulation is ideal for all laboratories including mobile laboratories or those in remote locations where cold storage may be limited.

The master mixes are produced in a certified laboratory manufacturing environment to the highest standards and are suitable for use on all qPCR machines. ROX is included - for use on platforms that require this as a passive reference dye. Each ampule of master mix can be used for 50 20 μL reactions, and each kit can be used for 150 20 μL reactions.

  Lyophilized Master Mixes
No need for dry ice or cold shipping
Simple protocol
Can be stored at ambient temperature
Stable for 12 months
Exceptional data quality
No loss of activity on repeated freeze-thaw cycles
Can be used in remote locations or away from a laboratory with limited cold storage capabilities
Suitable for use on all qPCR machines
Manufactured to highest standards
Hot start polymerase
Armored RNA Quant molecular controls banner