Real-Time Relative RT-PCR: How It's Done
Real-time PCR is performed on both the experimental samples and reference standards. Relative values for target abundance in each experimental sample is extrapolated from the standard curve generated from the reference standard. While the absolute values calculated for the experimental samples are meaningless, the relative differences in mRNA abundance between samples are accurate. When analyzing numerous samples, one sample is typically designated as the "calibrator" (or 1X sample), and the relative expression levels of all other samples are then expressed relative to the calibrator sample.
Improving Data Reliability by Including an Exogenous Control
To perform real-time RT-PCR analysis with an invariant control, a second set of PCR reactions is performed for the invariant endogenous control on both the reference dilution series and experimental samples. Relative abundance values are then calculated for 18S rRNA as well as for the experimental sequence. For each experimental sample, the relative abundance value obtained is divided by the value derived from the control sequence (18S rRNA) in the corresponding PCR. The normalized values for different samples can then be directly compared as described above.
A Real Life Example
|a2 M||18S||a2 M/18S||Value|
|Sample 1: kidney||82||3592||0.023||1.0|
|Sample 2: liver||18351||8966||2.05||90.0|
|Sample 3: ovary||44||1669||0.03||1.1|
|Sample 4: spleen||1||8||0.13||5.6|
Table 1. Relative Abundance of alpha-2 macroglobulin in Mouse Tissues Determined by Real-time RT-PCR.
Figure 1. Real-time RT-PCR Standard Curves. Standard Curves for alpha-2 macroglobulin and 18S rRNA derived from a dilution series of a reference RNA by real-time RT-PCR.
In addition to the optimized 10X real-time reaction buffer, SuperTaq Real-Time includes a separate tube of ROX Internal Standard (for TaqMan and SYBR Green chemistry on the ABI and Stratagene real-time instruments), 2.5 mM dNTPs, and 25 mM MgCl2. The Real-Time Reaction Buffer and ROX Standard are also available separately for customers who wish to use their current Taq polymerase and still realize substantial cost savings with Ambion's economically priced real-time reaction components.