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10 Tips for Successful Real Time qPCR

Enzo Life Sciences now provides 25 years of experience in the supply of research kits, biochemicals and biological. As Scientists Enabling Scientists, Enzo Life Sciences realizes the value in providing relevant information to our customers working in the fields of Life Science and Drug Discovery. We are happy to provide simple but useful tips for improving daily tasks as well as the overall quality of your research. With this in mind, here is a list of tips for achieving high quality data by quantitative PCR (qPCR) in real time, a method allowing the observation of specific DNA amplification as it is happening and the quantification of gene expression in a given sample.


1. Beware of the risks of contamination and cross-contamination

qPCR is a very sensitive way of measuring gene expression and therefore can be easily biased by contamination. That is why it is critical to obey the three-room rule for extraction, set-up and qPCR cycling. Gloves should be worn at all times and changed regularly. Aerosol-resistant pipette tips should also be used.



2. Small errors or deviations to the protocol will be automatically amplified

Pipettes should be calibrated regularly and used accurately for low volumes. Whenever possible, an already prepared qPCR mix should be used for better distribution of reagents between samples. If not, we suggest mixing the reagents thoroughly before use.


3. Primer integrity and quality are paramount for the generation of high quality data by qPCR

Primers should be stored in the appropriate buffer. DEPC-treated water may be slightly acidic and that is why it is important to neutralize it for maximum stability. A buffer with a neutral pH containing 1M EDTA to inhibit DNase is a viable alternative. At this concentration, EDTA should not interfere with the activity of Taq polymerase. Freeze/thaw cycles can lead to primer contamination and degradation. Primers should not undergo more than three to five freeze/thaw cycles. The standard operating procedure should be to work with 100-200mM stock solution and 10-20mM working solutions.


4. The previous statement is also true for RNA

Whenever possible, samples should be stored with a carrier or in low retention tubes. On the day of the experiment, freshly diluted material should be preferred. RNA quality and purity can affect reverse transcription efficiency and should be assessed using a micro-volume UV-Vis spectrophotometer. It should also be free of contaminating genomic DNA. We suggest performing a no-reverse transcriptase reaction to confirm absence of genomic DNA in your RNA samples. Designing forward and reverse primers on different introns is a viable alternative.


5. Optimization of qPCR reaction should be considered for each new target

For every new set of primers, it is critical to first check their efficiency. A standard five-point curve with 10-fold dilutions should help with that. The PCR efficiency with control DNA should be between 90 and 100%. Optimization of PCR parameters (e.g. reverse transcription and PCR primer design, template quality and quantity, cycling parameters including annealing temperature, and reaction buffer composition in magnesium, enzyme and other PCR enhancers) might be required.


6. Determining the optimal amount of RNA per reaction is a pre-requisite for successful quantitation

With qPCR, less is actually more. It is very often better to dilute the template and get Ct values above 15 cycles for accurate measurements of expression levels. A good starting point to measure the unknown expression levels of a gene is 100ng of total RNA template per reverse transcription reaction. The amount of RNA required for the reaction will be decided by the amount of target in each sample and can vary from 10pg to 100ng for high-copy and low-copy number RNA, respectively.


7. Fluorescent DNA-binding dyes versus probe-based qPCR approaches

The measurement of expression levels with fluorescent DNA-binding dyes does not differentiate between specific and non-specific amplicons. qPCR should be followed by melting curve analysis, which allows the confirmation of amplicons homogeneity. The presence of a single narrow peak in your melting curve will indicate specific amplification. Conversely, a single broad peak or multiple peaks will signify amplicons heterogeneity. Probe-based qPCR relies on an amplicon-specific probe capable of generating a fluorescent signal upon enzymatic process and amplification. The signal produced is specific of the target template. However, amount of probe in relation to the amount of primers and the amount of the target template will have to be optimized for accurate quantitation.


8. One-step versus two-step reverse transcriptase qPCR (RT-qPCR)

One-step qPCR is done sequentially in a single tube containing the RNA template. It is especially useful when working with very low amounts of RNA material. It is more sensitive than two-step qPCR and less cumbersome. Two-step qPCR is also performed sequentially but in different tubes. Only a small fraction of the RT reaction is used for qPCR allowing multiple PCR reactions from a single RT reaction. Two-steps qPCR are usually preferred when trying to quantify multiple targets with several replicates.


9. Check and double-check the program on the qPCR cycler

Your samples are now ready to be run in your qPCR cycler and it could be tempting to start the cycler immediately. However, several researchers might have had access to the instrument you are using and the program you are about to use might have changed dramatically. We, therefore, advise verifying that the program you are about launch is unchanged.


10. Your PCR experiments need to obey certain rules for publication

Stephen Bustin from Queen Mary University in London (UK) published several reviews on PCR and qPCR. With the help of colleagues, he collated back in 2009 a list of minimum information for publication of quantitative real time PCR experiments, also referred to as the MIQE guidelines. We strongly suggest going through this paper before submitting your qPCR data to a scientific journal. The information provided in your research article should be sufficient for readers and reviewers to rate the quality of your qPCR work and for other scientists to reproduce your data.

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References:

  • S.A. Bustin, et al. The MIQE guidelines : minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. (2009) 55: 611-22.

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