In this final installment on how to improve your qPCR gene expression profiling (see 6 Tips to Improve qPCR: part 1 and part 2 for previous discussion), we will discuss pipetting tips and the benefits of a separate reverse transcription step in qPCR template preparation.
Tip #5. Good Laboratory Practice (GLP) with pipetting helps to improve qPCR accuracy.
qPCR is a highly sensitive means for gene expression analysis because it amplifies its template exponentially, however, any deviations in each reaction are also amplified. A major source of such deviations is pipetting error. What can you do to prevent these deviations?
A good place to start would be by using a master mix, or a mixture of common reaction reagents such as DNA template, polymerase, qPCR dye and primers, instead of adding each reaction component individually. Using a master mix can significantly lower the frequency of pipetting and reduce pipetting error. To account for well-to-well variation using an internal control, an inert fluorescent dye, such as ROX™ Passive Reference Dye, can be included in the master mix as a loading reference, which allows for the normalization of pipetting variations.
Using appropriate pipettors which have been properly calibrated can also help. Each pipettor has an indicated volume range and should not be used above or below that range. For example, to dispense 1.5 μl, you can use a P2 pipette with 0.2-2 μl range, or a P10 pipette with 1-10 μl range, but not a P20 pipette with 2-20 μl range.
Finally, keep tip retention in mind. Tip retention refers to any reagent that is retained on both the outside and inside of the tip. It may seem insignificant, but a typical qPCR reaction volume is only 10-20 μl, and in this scale tip retention can affect qPCR accuracy. This is particularly true when you are dealing with small volumes such as 0.5 ng of cDNA template. To avoid introducing reagents retained on the outside of tips into the reaction, try to immerse only the very end of tips into the solution. To eliminate the impact of reagent loss from inside the tip, pre-wet the inside of tips by pipetting up and down a couple of times with the reagent to be transferred and always wait an extra second after dispensing the reagent. To minimize the influence of tip retention, consider using low retention tips.
Tip #6. Use a separate reverse transcription step to optimize your qRT-PCR.
To perform gene expression analysis from cell pellet samples by quantitative reverse transcription PCR (qRT-PCR), there are two options: one-step or two-step qRT-PCR. In one-step qRT-PCR, the RT step (for cDNA synthesis from total RNA) and PCR step are performed sequentially in the same tube, and all cDNA from the RT step is used in the PCR step as template. In two-step qRT-PCR, the RT step is separate and you can select the appropriate amount of cDNA to be used in the following PCR step. While one-step qRT-PCR may seem tempting because it requires less pipetting, we recommend two-step qRT-PCR for a number of reasons. First, with a separate RT step for cDNA synthesis, it is easier to control the cDNA amount to be used, a key factor for a successful qPCR. Too little cDNA and the detection threshold is not reached, yet too much cDNA may inhibit the qPCR reaction. Furthermore, a Cq value (the number of cycles when a qPCR amplification can be first detected) lower than 15 may interfere with the qPCR instruments default baseline settings. Second, with the two-step qRT-PCR it is easier to diagnose the source of an issue and correct the problem, rather than having to repeat the whole procedure. Finally, the extra cDNA saved can be used for other purposes such as quantifying extra genes, replicating assays, performing quality control or even cloning genes.
The third installment of the blog “6 Tips To Improve qPCR” concludes our trilogy. Though powerful and widely-used, attaining reliable results from qPCR can be difficult. We hope that our tips for primer design, wet-lab operation, and data analysis help you to obtain the trustworthy data you require.
Also, consider using our GeneQuery™ array kits and primers for your gene expression analysis needs. Our wet-lab validated primers can help save you precious time and effort. Our Ph.D. scientist team here at ScienCell is always ready to help. For more information, please visit http://www.sciencellonline.com/products-services/gene-expression-profiling.html, or email email@example.com.
Yongjiang Daniel Li, PhD
Research Scientist, Research and Development
ScienCell Research Laboratories
6076 Corte Del Cedro
Carlsbad, CA 92011
Ph: (760) 602-8549 ext.257