Can I switch to another qPCR master mix during an experiment?

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 .

Which is the best amplicon length?

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 type Optimal amplicon length
TaqMan / Hydrolysis probes 80 – 100 bp
Probes up to 100 bp
SYBRGreen / EvaGreen 120 – 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

When do I use TaqMan / Probes and when is SYBRGreen the best choice?

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

Is EvaGreen superior to SYBRGreen?

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

What is ROX?

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.

Why do I need a passive dye such as ROX?

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.

How do I test the function and reliability of a qPCR?

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)
How to optimise a qPCR

How do I optimise my 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
How do I optimise my qPCR?

Do plates and seals influence the qPCR assay performance?

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

Can I also analyse miRNA and lncRNA expression using qPCR?

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
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