Appropriate controls for qPCR experiments

April 21, 2011 09:51 Posted by David Vaudry

Controls are ideally required at every step of the PCR. Negative controls are used to detect nonspecific amplification, contamination or probe degradation. Positive controls are used to check the integrity of the sample, the stability of the reagents and the presence of inhibitors in the reaction mix.

The simplest and most important test is the No Template Control (NTC) which contains water in the reaction mix instead of the DNA material. This test is used to detect primer dimers and contamination. With SYBR Green, you might observe in some cases a late amplification due to primer dimerization. The lack of specificity of the signal can be confirmed on the dissociation curve. Nevertheless, this amplification should occur several cycles after the specific signal to avoid any misinterpretation. The specificity of the assay can be increased using a probe instead of the SYBR Green reporter dye.

Another important test is the no Reverse Transcriptase test (RT-) where the template is the RNA of the sample of interest prior to reverse transcription. Amplification in the RT- which is not detected in the NTC sample, strongly suggests the presence of contaminating genomic DNA. Contaminating genomic DNA can often be removed by DNAse treatment but the best way to avoid interferences with genomic DNA is to design intron spanning primers or intron flanking primers.

When using a probe in your assay, you can also mix all reagents excepted the polymerase to check for the integrity of the probe. If in such conditions, an increase of the fluorescence signal during the PCR process, suggests that the probe gets degraded. Probe degradation may also be suspected when a high background signal is observed.

An endogenous positive control is a validated well known set of primers targeting a specific gene which is analyzed at the same time as the gene of interest. It can be run in multiplex but also often on contiguous wells. Endogenous positive controls give information regarding the quality of the buffer and/or activity of the polymerase. Endogenous positive controls can be the reference genes which are used to correct for sample-to-sample variations and to normalize the results for the gene of interest. It is possible to design several sets of primers (3 to 5) targeting different regions of the endogenous positive controls in order to identify possible degradation of the matrix.

An exogenous positive control is a different DNA template in which the gene of interest is known to be expressed (it can be RT product, PCR product or plasmid).This control can be used to check for the quality of the primers, probe and reagents but won’t give information regarding the sample (degradation or presence of inhibitors). It is possible to make your own controls but some commercial qPCR Control kits are also available.

When the presence of inhibitors is suspected, an Internal Positive Control (IPC) can be added to your sample. An IPC is a known amount of DNA or RNA that is mixed with the sample and amplified with specific primers. Any variation in the expression level of the IPC indicates the presence of inhibitors in the sample. For more information regarding appropriate controls for inhibition, click here.

For more information regarding appropriate controls for qPCr experiments, click here.