Real Time and qPCR

Quantitative PCR assays

Most people use Sybr-green assays to perform quantitation by PCR at our facility. This assay uses a double stranded DNA binding dye which fluoresces only in the presence of dsDNA. The Sybr-green assay is economical and sensitive, but requires highly specific primers. If non-specific PCR products are produced, quantitation will not be possible with Sybr-green because the dye will bind to any product as well as primer di-mers.

Alternately, one can use Taqman assays in which a third primer is used in addition to the traditional PCR primer pair. This third primer, called a probe, is labeled with both a fluorescent molecule and a quencher molecule. It binds to the target sequence between the two PCR primers. As the reaction proceeds, the exonuclease activity of Taq enzyme cleaves the probe, liberating the fluor and quencher molecules. Since the fluor and quencher are no longer in close proximity fluorescence is no longer quenched, and the fluorescent signal is then collected by the RealTime instrument. The advantage to this type of assay is that more than one gene may be monitored in a single reaction (i.e. fluors with different emission wavelengths may be used together). Multicolor detection allows a control gene (usually a constitutively expressed gene whose expression does not vary in response to environment) to be amplified along with the gene of interest, simplifying the analysis phase of the experiment.

The drawback to using multicolor detection is that it can be expensive. Our facility usually recommends that studies involving large numbers of experimental data points for only a few genes will benefit from Taqman assays. On the other hand, studies in which a large number of genes must be validated among a small number of RNA samples will be more economical when using Sybr-green detection.

Below are some additional points for those considering quantitative PCR.

You will need special plasticware with optical quality covers. These are available from several distributors such as Applied Biosystems, Axygen, and Bio Rad. The Applied Biosystems plasticware is designed to be most compatible with the ABI 7000.

The following plasticware is recommended:

  • 96-well optical plates (Fisher Scientific #AB-1400)
  • 384-well optical plates (Fisher Scientific #AB-1310)
  • optical adhesive covers (Fisher Scientific #AB-1170)

or

  • optical tube strips (Applied Biosystems cat#4316567)
  • optical strip caps (Applied Biosystems cat#4323032)

Master mixes/enzymes:

When doing quantitative PCR, the first step is to make cDNA. We have found that the Superscript III reverse transcriptase (Invitrogen cat#18080-044) works well. There are also first strand synthesis kits that have primers (oligo dT and/or random primers) already in the master mix, such as the Bio-Rad iScript cDNA synthesis kit (cat #170-8890), among others. If using gene-specific primers to prime the cDNA reaction, as well as the PCR reaction, a one-step kit may be used. This is a convenient approach because all of the reactions may be done in the same tube. No purification of the cDNA template is necessary prior to PCR. Please contact the UAGC for more information on these kits.

When using the ABI 7300 or 7900 instrument, it will be necessary to use a qPCR master mix that contains a ROX passive standard. Using a ROX fluorescent standard will correct for variability in excitation light source levels from well to well. There are several such master mixes. Below are just a few which can be used with Sybr-green detection assays. Feel free to contact us for information on master mixes for use with Taqman probe assays.

The following master mixes are recommended:

  • Sybr-Green PCR Master Mix (Applied Biosystems cat #4309155)
  • Bio-Rad iTaq Sybr green supermix with ROX (Bio-Rad cat #170-8850)

Sybr Green primer design:

Due to the fact that Sybr-green binds to all double stranded DNA, it is extremely important to have a good primer pair which does not form dimers. It is best to avoid using primer-pairs that create dimer structures with DG higher than 6kcal. There are several good web based primer design programs. Our facility recommends Primer3 for its simplicity of use. Commercially available programs such as Oligo work very well also.

Dual labeled probe design:

The University of Arizona Genetics Core has Applied Biosystems™ Primer Express software for those interested in Taqman probe design. This software works well. However, the Assays-by-design service from ABI can save time and effort. For a fee, ABI can design primers and probes for your gene of interest. They will functionally test the reactions, and send you a master mix ready for use. Feel free to contact our facility for further information.

Normalizion techniques:

Variability in RNA template concentrations will make quantitation by PCR inaccurate. There are two common ways to control for variations in total RNA levels in each RT reaction. The first is to use a control gene like B-actin or GAPDH to act as a loading control. Sometimes, the use of a housekeeping gene is not possible, in which case RNA concentrations need to be standardized. The most sensitive and accurate means of measuring RNA is with RiboGreen reagent from Molecular Probes. You will need a fluorometer to use this system. Also, a UV spectrophotometer may be used.

Data replication:

It is always good practice to replicate data points at least three times for any experiment. The sensitivity of qPCR makes this practice a necessity. Inaccuracies in pipetting and RNA quantitation will invalidate quantitative PCR results if not compensated for. When replicates are performed, standard deviations will reveal the reproducibility of the data.

PCR efficiency:

It is VERY important to know the efficiency of your quantitative PCR reaction. If the reaction is not proceeding at 100% efficiency, then there will not be a doubling of PCR product in each cycle. Fold change calculations rely on the assumption that there is a doubling of product per cycle. Two reactions may be directly compared to each other, if they are operating at the same efficiency (preferably above 90%). However, if the control and gene of interest are amplifying with different efficiencies it is not recommended that they be directly compared.

There is a mathematical technique for correcting for PCR amplicons of different efficiencies presented by M. W. Pfaffl (A new mathematical model for relative quantification in real-time RT-PCR, Nucleic Acids Research, 2001, Vol. 29, No. 9 Article number e45). Please contact the University of Arizona Genetics Core facility if you need further information on the subject.