Three common problems and solutions to getting reproducible qPCR results!
Real-time polymerase chain reaction (qPCR) is an extremely sensitive assay in which the slightest variation can influence the results. Thus, to have a consistent and efficient qPCR assays, several parameters must first be optimized or taken into account. Below are three common problems and solutions to getting reproducible qPCR results:
- Problem: Inconsistent results
- Consistent Cell Growth: Due to the reliability and sensitivity of qPCR assays, this assay has been adopted as the method of choice for a wide range of applications including molecular diagnostics, genotyping, and gene expression studies. For the latter assay, the health of the cells or sample can have a tremendous effect on the variability or reproducibility of the results. As such, the pattern and timing of gene expression can vary from experiments to experiment if the condition of cell growth causes cell stress or inconsistency in growth. One way to have a consistent growth is to use quality tissue culture ware such as our TPP dishes and plates. Our line of TPP products are made from the highest purity plastics and are innovatively modeled to improve generation of consistent and reproducible data. These dishes offer a flatter, more consistent growth surface, even temperature distribution, and reduced evaporation by having stacking air vents. Try out a sample and see for yourself.
- Evaporation problems: It is important to make sure that your plates or tubes are properly sealed in order to prevent evaporation. If the event of evaporation, the signal from the reporter dyes will increase due to an increase in signal concentration which will influence the assay results. Depending on the extent of the evaporation, you may get useable results but there will be variation from assay to assay. If you are experiencing evaporation, here are some way to minimize it: Make sure the right cap or sealing film is paired with the right plate and tubes, (2) make sure the seal properly covers all wells including the edges of the plate where evaporation most often occurs, and (3) use a roller or straight edge to thoroughly seal the adhesive cover to the plate. Find the right sealing film for your PCR needs here: http://shop.midsci.com/scategory/M50/929
- Use quality chemical and consumables: Due to the sensitivity of the assay, the slightest mistake in reaction composition or pipetting can throw off the final results. It is for this reason that most use ready-to-use reaction master mix to minimize the number of pipetting steps, laminar flow clean bench, calibrated pipette, and low binding barrier-tips to prepare their samples. It is also best to use low-binding plastic-ware (PCR tubes, strips, and tips) to minimize sample loss and increase consistency across samples and experiments. Also, the use of barrier tips and clean benches reduce the chance of contamination.
- Problem: No signal or low signal
- Use quality DNA template: The old adage “garbage in garbage out” applies. This is why several control reactions are included as quality control check for every reaction. Be sure to use quality DNA extraction and reverse transcription kits for a clean sample preparation, free of any PCR inhibitors. Imperfect purification of nucleic acid can leave trace amount of substance that can inhibit or interfere with PCR reactions.
- Use validated primer and probe sequence: There are several tools available online that can assist with assessing primer and probe properties. Be sure to check the literature (and RTPrimerDB database) for validated or published probe and primer sequences. Be sure to also follow best practices when it comes to choosing target sequences such as avoid using regions having secondary structures, single nucleotide repeat regions (>4), and high GC content regions; aim to amplify a short region (75-200bp), because short sequence are amplified with higher efficiency, but long enough for the product to be distinguish from any primer-dimer formation.
- Adjust the sensitivity of detection in your instrument: Each instrument has a specific sensitivity and dynamic range. Some instrument can detect as low as one copy of the gene while others are less sensitive. The sensitivity also effects the signal-to-noise ratio (ratio of specific hybridized fluorescent signal to nonspecific hybridization event) in which the higher the ratio, the greater the sensitivity of the instrument. Consequently, if you don’t see any peaks for your qPCR assay, then perhaps the threshold is set too high in which the lower peak signals are disregarded as noise.
- Problem: Errors in quantification
- a.Data Analysis: While qPCR assays offers high sensitivity (with a broad dynamic range of quantification) and reproducibility, it is important to have the proper controls in place for an accurate analysis and interpretation of the results. Quantification variation can result from a variety of sources such as DNA quality, reaction efficiency, enzyme inhibition, and many other factors. It is for this reason that an internal control is employed to compare the expression of the gene of interest in relation to another gene expression.
- b.Use a reference dye: If your instrument allows it, use a reference dye, such as ROX dye, to compensate for any well-to-well variation. Because the reference dye concentrations are constant during the PCR reaction, the dye signal should be the same for all wells. Although the use of a reference dye is not necessary, be sure you know how your instrument compensates for any well-to-well variations.
Lastly, if you are having difficulty with your qPCR assays or planning to start a project, let MIDSCI help you select the right qPCR enzyme for your specific thermal cycler, tubes and plates, sealing film, and barrier tips for successful and reproducible results.