Comparative Ct Method Another quantitation approach is termed the comparative C
t method. This involves comparing the C
t values of the samples of interest with a control or calibrator such as a non-treated sample or RNA from normal tissue. The C
t values of both the calibrator and the samples of interest are normalized to an appropriate endogenous housekeeping gene.
The comparative C
t method is also known as the 2
–[delta][delta]Ct method, where
[delta][delta]C
t = [delta]C
t,sample - [delta]C
t,referenceHere, [delta]C
T,sample is the C
t value for any sample normalized to the endogenous housekeeping gene and [delta]C
t, reference is the C
t value for the calibrator also normalized to the endogenous housekeeping gene.
For the [delta][delta]C
t calculation to be valid, the amplification efficiencies of the target and the endogenous reference must be approximately equal. This can be established by looking at how [delta]C
t varies with template dilution. If the plot of cDNA dilution versus delta C
t is close to zero, it implies that the efficiences of the target and housekeeping genes are very similar. If a housekeeping gene cannot be found whose amplification efficiency is similar to the target, then the standard curve method is preferred.
Instrumentation for Real-Time PCR Real-time PCR requires an instrumentation platform that consists of a thermal cycler, a computer, optics for fluorescence excitation and emission collection, and data acquisition and analysis software. These machines, available from several manufacturers, differ in sample capacity (some are 96-well standard format, others process fewer samples or require specialized glass capillary tubes), method of excitation (some use lasers, others broad spectrum light sources with tunable filters), and overall sensitivity. There are also platform-specific differences in how the software processes data. Real-time PCR machines are not inexpensive, currently about $25K - $95K, but are well within purchasing reach of core facilities or labs that have the need for high throughput quantitative analysis. For a comprehensive list of real-time thermal cyclers please see the weblink at the end of this article.
Tools for Real-Time RT-PCRAmbion’s
MessageSensor™ RT Kit includes an RNase H+ MMLV RT that clearly outperforms MMLV RT enzymes that have abolished RNase H activity in real-time RT-PCR experiments. Unlike many other qRT-PCR kits, MessageSensor includes a total RNA control, a control human GAPDH primer set, RNase inhibitor, and nucleotides, as well as a buffer additive that enables detection with SYBR® Green dye.
The
Cells-to-cDNA™ II Kit produces cDNA from cultured mammalian cells in less than 2 hours. No RNA isolation is required. This kit is ideal for those who want to perform reverse transcription reactions on small numbers of cells, numerous cell samples, or for scientists who are unfamiliar with RNA isolation. Ambion's Cells-to-cDNA II Kit contains a novel Cell Lysis Buffer that inactivates endogenous RNases without compromising downstream enzymatic reactions. After inactivation of RNases, the cell lysate can be directly added to a cDNA synthesis reaction. Cells-to-cDNA II is compatible with both one-step and two-step real-time RT-PCR protocols.
Genomic DNA contamination can lead to false positive RT-PCR results. Ambion offers a variety of tools for eliminating genomic DNA contamination from RNA samples prior to RT-PCR. Ambion’s
DNA-free™ DNase Treatment and Removal Reagents are designed for removing contaminating DNA from RNA samples and for the removal of DNase after treatment without Proteinase K treatment and organic extraction. In addition, Ambion has also developed
TURBO™ DNase, a hyperactive enzyme engineered from wild-type bovine DNase. The proficiency of TURBO DNase in binding very low concentrations of DNA means that the enzyme is particularly effective in removing trace quantities of DNA contamination.
Ambion now also offers an economical alternative to the high cost of PCR reagents for the ABI 7700 and other 0.2 ml tube-based real-time instruments.
SuperTaq™ Real-Time performs as well or better than the more expensive alternatives, and includes dNTPs and a Reaction Buffer optimized for SYBR Green, TaqMan, and Molecular Beacon chemistries.
End-Point RT-PCR: Relative vs. Competitive vs. Comparative
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