Real Time PCR

Overview

 Real-time PCR is one of the services available in the DNA Chemistry Core of the Biomolecular Research Facility. Using the ABI Prism® 7900HT Sequence Detection System, we are able to analyze RNA and DNA samples for the purpose of determining relative quantitation of gene expression. In addition to quantifying gene expression, potential applications for real-time PCR include mutation detection, allelic discrimination, and detection of single nucleotide polymorphisms (SNPs) using the fluorogenic 5' nuclease assay.

TaqMan Chemistry and Instrumentation

TaqMan probes contain two dyes, a reporter dye (e.g. 6-FAM) at the 5’ end and a quencher dye (e.g. Black Hole Quencher) at the 3’ end. The proximity of the quencher dye to the reporter in an intact probe allows the quencher to suppress, or “quench” the fluorescence signal of the reporter dye through Förster-type energy transfer. If the target of interest is present, these probes specifically anneal between the forward and reverse primer sites. During the reaction, the 5’ to 3’ nucleolytic activity of the AmpliTaq Gold enzyme cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are displaced from the target, separating the reporter dye from the quencher dye and thus resulting in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. Because increase in fluorescence signal is detected only if the target sequence is complementary to the probe, nonspecific amplification is not detected.

The 7900HT is currently optimized to detect 8 dyes:
reporter dyes, FAM™, TET™, JOE™, VIC™, SYBR® Green;
the fluorescent quencher TAMRA™;
non-fluorescent quenchers, such as Black Hole Quencher™ ;
and passive reference ROX™ . 
The passive reference dye does not participate in the 5’ nuclease PCR, but instead provides an internal reference to which the reporter-dye signal can be normalized during data analysis.   This is necessary to correct for fluorescent fluctuations due to changes in concentration or volume in the wells.  Normalization is accomplished through ABI’s Sequence Detection System (SDS) software, which divides the emission intensity of the reporter dye by the emission intensity of the passive reference to obtain a ratio defined as the Rn  (normalized reporter) for a given reaction well.  The difference between the Rn value of a reaction containing all components including the template (Rn+), and the Rn value of an unreacted sample (Rn-) equals the ΔRn value, which reliably indicates the magnitude of the signal generated by the given set of PCR conditions.

Design of TaqMan Primers and Probes

A successful run of real time PCR relies heavily on the primers and probes and the conditions of the PCR. Specificity is always of primary concern when designing the primers and probes. Several databases -public or private-, web tools and commercial programs, such as ABI’s Primer Express,  are available to help one make the selections.

Some guidelines for TaqMan probes and primers selection are as follows:

  • G-C content between 20% and 80%
  • Avoid runs of an identical nucleotide, especially guanine
  • Avoid G to be on the 5’ end
  • Probes and primers should contain more C than G
  • Melting temperature (Tm) should be 68-70°C for probes and 58-60°C for primers
  • The five nucleotides at the 3’ end of each primer should have no more than two Gs and/or Cs
  • Give precedence of better probes over primers
  • Primers should be as close to probe as possible without overlapping

Alternative Chemistries

Other capabilities of the ABI Prism™ 7900HT are still being explored. We are currently looking into the use of SYBR® Green, which does not require a fluorescent probe, can be a desirable option, especially for assays involving a large number of genes. A typical example of such applications is verification of a microarray experiment

Data Analysis
The standard curve method is used to determine relative quantitation.  Absolute quantitation may also be calculated using this method, but this requires that the absolute quantities of the standard be known by some independent means.  The most important parameter for quantitation is the threshold cycle (CT) value.  This value indicates the cycle at which a statistically significant increase in ΔRn is first detected.  Usually, this occurs when the Signal Detection Software begins to detect the increase in signal associated with an exponential growth of PCR product.  The SDS software then generates a standard curve plot with the log (ng) input amount of RNA in each well as the X values and CT as the Y values. Local Image 

Standard curves are prepared for both the target and an endogenous reference such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or 18S amplicon ribosomal RNA (rRNA).  For each experimental sample, the amount of target and endogenous reference is determined from the appropriate standard curve.  Then, the target amount is divided by the endogenous reference amount to obtain a normalized target value.  One of the experimental samples is selected as the calibrator, or 1x sample.  Each of the normalized target values is divided by the calibrator normalized target value to generate the relative levels of expression.  Thus, the normalized amount of target is a unitless number and all quantities are expressed as an n-fold difference relative to the calibrator.

Other capabilities of the ABI Prism™ 7900HT are still being explored.  We are currently looking into the use of SYBR® Green, TaqMan® MGB probe and primers, and multiplex PCR.

If you wish to use this service, contact  Yongde Bao  at 982-2551 or 924-2553. Information on samples should be supplied on the rt-pcr form which can be completed on a computer but must be printed; data is not transmitted electronically.

Our instrument

Local Image