qPCR FAQ

Frequently Asked Questions (FAQ) qPCR

In this section you will find answers to the most common questions around qPCR. If you have any further questions or comments please do not hesitate to contact us directly via phone or email.

More and more researches focus on microRNA (miRNA) and long non-coding RNA (lncRNA) as they seem to play an important role in gene regulation. QPCR is a method also suited to quantify miRNA and lncRNA (Lui 2013, Jiang 2014) and with primaQUANT miRNA we  have designed a special qPCR product for you.

For more detailed information about the use of qPCR for miRNA oder lncRNA analysis please see:

Liu, S.-P., Yang, J.-X., Cao, D.-Y., & Shen, K. (2013). Identification of differentially expressed long non-coding RNAs in human ovarian cancer cells with different metastatic potentials. Cancer Biology & Medicine, 10(3), 138–141. https://doi.org/10.7497/j.issn.2095-3941.2013.03.003
Jiang, X.-Y., & Ning, Q.-L. (2014). Expression profiling of long noncoding RNAs and the dynamic changes of lncRNA-NR024118 and Cdkn1c in angiotensin II-treated cardiac fibroblasts. International Journal of Clinical and Experimental Pathology, 7(4), 1325–1336. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/24817929
Mou, G., Wang, K., Xu, D., & Zhou, G. (n.d.). Evaluation of Three RT-qPCR-Based miRNA Detection Methods Using Seven Rice miRNAs. https://doi.org/10.1271/bbb.130192
Wan, G., Lim, Q. E., & Too, H.-P. (2010). High-performance quantification of mature microRNAs by real-time RT-PCR using deoxyuridine-incorporated oligonucleotides and hemi-nested primers. RNA (New York, N.Y.), 16(7), 1436–1445. https://doi.org/10.1261/rna.2001610
Androvic, P., Valihrach, L., Elling, J., Sjoback, R., & Kubista, M. (2017). Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Research, 45(15). https://doi.org/10.1093/nar/gkx588

It is a myth that one cannot switch to another master mix within a project or series of experiments! The only prerequisit to switch to a better product is that you have normalised your assay. (Which one should do anyway).

Our recommendations for a successful switch:

  • Run your negative and positive controls with both mixes in parallel. This allows you to directly compare their performances.
  • Have a close look at the fluorescence curve –  are there signs for a PCR inhibition?
  • Run your assay with a cDNA dilution series to compare the dynamic range of different master mixes
  • Check if the setup of your cycler fits the requirements of the master mixes tested

For further information please visit our qPCR Knowledge Center – How to test and optimise a qPCR .

Melting curves are an essential tool to estimate the specifity of a qPCR. You can instantly see if the right target has been amplified, or if unwanted by-products are being produced and detected. We strongly recommend to perform a melting curve analysis to be sure that the desired target has been amplified!

The cycler creates the melting curve by measuring the fluorescence while constantly raising the temperature. As soon as the temperature exceeds the melting temperature of the amplicon, it becomes single stranded resulting in a sudden drop of the fluorescence.

Please be aware that the peak of the melting curve may slightly vary between different master mixes. No reason to worry, as long as there is only the one peak to be expected! If the curve shows several peaks – exception multiplex assay – you cannot really trust the results. Further investigation is necessary to evaluate the reason.

A melting curve analysis requires intercaling dyes like e.g. SYBRGreen oder EvaGreen and is not possible in a probe assay.

Plates and seals may well influence the performance of your assay (Reiter 2008).
White assay plates like e.g. our primaPLATE SL-PP384-LC oder 4ti-0951 generally show a higher sensitivity due to a reduced light distraction compared to standard clear plates.

The sealing film has also a considerable effect for the performance. You should thus not use standard PCR adhesive films, but optically clear seals instead. You can choose from pressure sensitive films, strong adhesive films, or heat-sealing films.

Pressure sensitive films like our primaSEAL qPCR-2 are non-sticky and need a certain pressure for sealing. Heat-sealing films are first choice if you need maximum sealing. Although this option requires a heat sealing device, it may also pay off financially if you constantly have a large amount of plates to seal.

Reiter, M., & Pfaffl, M. W. (2008). Effects of Plate Position, Plate Type and Sealing Systems on Real-Time PCR Results. Biotechnology & Biotechnological Equipment, 22(3), 824–828. https://doi.org/10.1080/13102818.2008.10817561

The following parameters determine the specifity of a qPCR assay

  • Primer concentration
  • Annealing temperature and the melting temperature of the primers
  • Annealing time
  • Occurence of primer-dimer

What can you do if your qPCR shows a lack of specifity?

  • Decrease the primer concentration

  • Increase the annealing temperature – but do not exceed the melting temperature of the primers!

  • Reduce the annealing time. That makes the annealing of the primers more stringent so that primers with the best match mostly make the race.

  • Design new primers with higher melting temperature and optimal length.

Check the specifity of your qPCR via agarose gel, or – in case you use SYBRGreen – via the melting curve.

It is very important to determine the primer efficiency for each gene to be analysed.

Make a 10-fold dilution series of 5-6 steps and generate a graph with the results. Next step is to calculate the slope of the data series plot which allows to calculate the efficiency. It should figure between 85% and 110%.

For more detailed information please see section “How to optimize a qPCR”.

The optimisation of a qPCR needs several steps:

  • Check for PCR inhibitors
  • Calculation of the primer efficiency
  • Estimation of the Linear Dynamic Range (LDR)
  • Optimisation of the primer concentration
  • Optimisation of the primer quantity
  • Optimisation of the consumables (plates, seals, pipet tips)
  • Optimisation of the size of the amplicon
  • Selection of the best-working qPCR master mix

There are different parameters to consider to end up with an optimised qPCR assay.
The MIQE Guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Bustin 2009) provide a useful overview. To make it more convenient, we have put together the most important factors in an extra guide for you:

How to estimate and compare the performance of a qPCR assay

Here in short:

  • Does the quality of my RNA match? (FragmentAnalyzer, Bioanalyzer, NanoDrop curve)
  • Test the primer efficiency with a dilution series
  • Does my amplicon match with the linear dynamic range of my assay? (5-6 step 10-fold dilution series)
  • How high is the variability between my replicates  and biological samples?
  • Does my assay produce primer-dimer or other by-products? (melting curve analysis)

There are many factors to be considered – and to be tested – to answer this question.

Please be aware that too much template as well as products of the reverse transcription process may inhibit the qPCR reaction. We thus strongly recommend to start with a 5-6 step 10-fold dilution series (e.g. 0.001 ng, 0.01 ng, 0.1 ng, 1 ng, 10 ng) to determine the optimal amount of template for your setup.

A dilution series allows to determine the following parameters:

  • Efficiency of primers
  • Inhibition of the qPCR reaction by template/rT-products
  • The amount of template needed to run the qPCR in the linear slope

It is important to adjust the amount of template in a way that the qPCR reaction runs in the linear part of the amplification curve. Typically this is the case with in between 1 ng and 10 ng template/reaction.

Besides SYBRGreen there are other intercalating dyes such as EvaGreen used for qPCR.

Normally, you should not find relevant differences in gene expressions between both dyes. However, EvaGreen might produce slightly more defined peaks during HRM (High-Resolution Meltinc Curve Analysis), (Eischeid 2011).

For standard gene expression assays SYBRGreen has proven excellent performance and sensitivity.

Additional informationen about melting curve analysis:

Eischeid, A. C. (2011). SYTO dyes and EvaGreen outperform SYBR Green in real-time PCR. BMC Research Notes, 4, 263. https://doi.org/10.1186/1756-0500-4-263

Though a qPCR assay mostly needs to be optimised individually, there are a few standard protocols which may be helpful to use at the beginning.

If you choose a ready-to-use master mix  like our primaQUANT mix, all you have to do is to add your primers and the cDNA template. With our qPCR Mastermix Calculator the calculation of the qPCR assay becomes a pretty easy task and takes only a few seconds.

For an overview of existing standard protocols in 96- and 384-format please click here.

TaqMan™ probes are hydrolysis-probes suitable for quantification analysis with qPCR.

Hydrolysis-probes are single stranded DNA oligos labeled with a fluorophore on one end and a quencher on the other end. The quencher surpresses the fluorescence unless the probe binds to an amplicon and the polymerase with its exonulease activity cuts off both labelings. Then a fluorescence will occur subsequently to a trigger beam and can be measured.

The amount of free fluorophores – and thus the fluorescence signal – is proportional to the amount of amplified DNA .

ROX (5-Carboxy-Rhodamin-X) is a fluorescence dye which some qPCR cyclers use as passive reference dye. This allows to mathematically eliminate non-specific fluorescence signals, resulting from well-to-well variances due to material inconsistencies or unprecise pipetting. So please use this normalisation option if your qPCR machine works with ROX and check which ROX level it requires. However, it is not a problem, if the ROX option is not available. If your qPCR assay is thoroughly validated, you will also get good results without ROX.

Please look here for a list of qPCR cyclers that require ROX.

The Ct (Cycle threshold) – or more correctly – Cq (Cycle quantification) – value  is per definitionem being used to determine the gene expression via the fluorescence. Basically, one determines mathematically (commonly used is the 2nd derivative-method) the value where the fluorescence does not increase any more which indicates that the exponential phase of the amplification has ended. This happens in a certain cycle which is then called the “quantification cycle” (Cq).

However, there are many more mathematical methods to determine the Cq-value (see literature below). Most important is that you always use the same calculation method within one experiment.

Ruijter, J. M., Zhao, S., Spiess, A. N., Boggy, G., Blom, J., Rutledge, R. G., … Vandesompele, J. (2013). Evaluation of qPCR curve analysis methods for reliable biomarker discovery: Bias, resolution, precision, and implications. Methods, 59(1), 32–46. https://doi.org/10.1016/J.YMETH.2012.08.011

The primer concentration has a major effect on the performance of a qPCR and must thus be optimised thoroughly. If the concentration is too low or too high, the calculation of the Cq-values may result incorrect, and the danger of primer-dimers increases. A good guess is to start with a primer concentration of 200-300 nM and to also include higher and lower concentrations in a test assay.

Mikeska, T., & Dobrovic, A. (2009). Validation of a primer optimisation matrix to improve the performance of reverse transcription – quantitative real-time PCR assays. BMC Research Notes, 2, 112. https://doi.org/10.1186/1756-0500-2-112

Several papers document that it is rather a myth than reality that an assay with hydrolysis probes (TaqMan®-assay) is superior to a SYBRGreen Assay  (Tajadini 2014, Cao 2012, Arikawa 2008). Provided that you design the right primer, use a high quality qPCR master mix like our primaQUANT and work with non-fragmented cDNA (same is true for TaqMan® assays as well).

Probe-based assays are especially suited for SNP-genotyping, for the analysis of splicing variants, and for the detection of mutations via qPCR.

If you are interested in gene expression or miRNA expression, SYBRGreen assays are to be preferred and allow additional quality parameters like melting curve analysis.

Our primaQUANT qPCR Mastermixes are available for SYBRGreen as well as for probes.

For more detailed information please see the following publications:

Tajadini, M., Panjehpour, M., & Javanmard, S. H. (2014). Comparison of SYBR Green and TaqMan® methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes. Advanced Biomedical Research, 3, 85. https://doi.org/10.4103/2277-9175.127998
Cao, H., & Shockey, J. M. (2012). Comparison of TaqMan® and SYBR Green qPCR Methods for Quantitative Gene Expression in Tung Tree Tissues. Journal of Agricultural and Food Chemistry, 60(50), 12296–12303. https://doi.org/10.1021/jf304690e
Arikawa, E., Sun, Y., Wang, J., Zhou, Q., Ning, B., Dial, S. L., … Yang, J. (2008). Cross-platform comparison of SYBR Green real-time PCR with TaqMan® PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC) study. BMC Genomics, 9, 328. https://doi.org/10.1186/1471-2164-9-328

We recommend to include – depending on the qPCR assay formate – the following controls:

  • No RT – control. If  you work with a reverse transcript from mRNA as template, you should include samples without enzyme as a noRT control.
  • No Template Control (NTC) – Run each gene with NTCs using water instead of template to check for cross contamination.
  • Reference genes (“housekeeping genes”). For each sample several reference genes should be included to enable a subsequent quantification. Please check upfront that the reference genes are not influenced by the intended treatment of your sample.  
  • Alternative Positive Control – For absolute quantification assays, in which no reference genes are used, an alternative positive control is required. This can include a control with a well defined amount of cDNA, that will show already known results.

The robustness, sensitivity, and specificity of a qPCR Assay also depend on the amplicon length. This is especially true for Taqman/probe assays, whereas a SYBRGreen assay reacts robustly towards different amplicon lengths as shown in fig 1a/b.

It is generally advised not to have different amplicon lengths within one assay.

Assay typeOptimal amplicon length
TaqMan / Hydrolysis probes80 – 100 bp
Probesup to 100 bp
SYBRGreen / EvaGreen120 – 200 bp

Fig 1a: Different amplicon lengths within one assay should be avoided

Mikeska, T., & Dobrovic, A. (2009). Validation of a primer optimisation matrix to improve the performance of reverse transcription – quantitative real-time PCR assays. BMC Research Notes, 2, 112. https://doi.org/10.1186/1756-0500-2-112

Fig 1b: Taqman and SYBRGreen assays require different amplicon sizes

Mikeska, T., & Dobrovic, A. (2009). Validation of a primer optimisation matrix to improve the performance of reverse transcription – quantitative real-time PCR assays. BMC Research Notes, 2, 112. https://doi.org/10.1186/1756-0500-2-112

Each cell line or assay requires a validation of reference genes for a robust gene expression analysis. Alternatively, one can try “classical” housekeeping genes as reference standard which have proven their suitibility as controls in a variety of assays.

On the right side you will find an overview of commonly used reference genes. For more details please see the following publications:

Ahrberg, C. D., & Neužil, P. (2015). Doubling Throughput of a Real-Time PCR. Scientific Reports, 5(1), 12595. https://doi.org/10.1038/srep12595
Gholami, K., Loh, S. Y., Salleh, N., Lam, S. K., & Hoe, S. Z. (2017). Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence. PLOS ONE, 12(6), e0176368. https://doi.org/10.1371/journal.pone.0176368
Radonić, A., Thulke, S., Mackay, I. M., Landt, O., Siegert, W., & Nitsche, A. (2004). Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications, 313(4), 856–862. https://doi.org/10.1016/J.BBRC.2003.11.177
Kozera, B., & Rapacz, M. (2013). Reference genes in real-time PCR. Journal of Applied Genetics, 54(4), 391–406. https://doi.org/10.1007/s13353-013-0173-x
Gong, H., Sun, L., Chen, B., Han, Y., Pang, J., Wu, W., … Zhang, T. (2016). Evaluation of candidate reference genes for RT-qPCR studies in three metabolism related tissues of mice after caloric restriction. Scientific Reports, 6(1), 38513. https://doi.org/10.1038/srep38513
HGNC Symbol
GAPDH
PGK1
ACTB
HPRT
TUBA
HMBS
GUSB
RPL13A
YWHAZ
Β2M
TBP
PPIA
RPII
G6PDH
ALB

ROX (5-Carboxy-Rhodamin-X) is a fluorescent dye which some qPCR cyclers use as a passive reference.

ROX is added to each reaction and then the ROX-signals of each sample (well) are being mathematically normalised. With this method well-to-well differences in fluorescence resulting from unprecise pipetting or material inconsistencies can be ruled out. This is basically a good approach and we thus recommend to use the ROX option if available. It is important to then use a master mix with the ROX concentration required by the cycler. However, no need to worry if your qPCR cycler does not work with ROX. ROX is nice to have, but not a must. A thoroughly established and validated assay works well without ROX.

Please see here for a list of cyclers which need ROX.

Your Product Specialist

Jan Winter

Product Manager Genomics

PCR, qPCR, Magnetic Beads & CRISPR/Cas9

+49 (152) 3361 2537

jw@steinbrenner-laborsysteme.de