Real-Time PCR analysis detects specific nucleic acid amplification products as they accumulate in real-time. The ABI Prism® 7900 Sequence Detection System from Applied Biosystems is used to monitor the increase of the reporter fluorescence during PCR. The substitution of the old 7700 by the 7900 Sequence Detection System allowed a decrease in the reaction price since it requires much lower quantities of reagents. Also, the improved software gives a much more efficient analysis.
Starting Your RT-PCR Experiment
Submit the cDNA sequence of the gene of interest
(eukaryotic or prokaryotic)
- Prepare your samples: ~1µg of DNA or RNA*
*Note: RNA samples must be free of DNA contamination
- Design the appropriate primers and probes,
including those for endogenous control
- Synthesize and purify the selected primers and TaqMan probes
- Set up the reactions for RT-PCR in 384-well plates
- Run the microplates in the ABI PRISM® 7900
- Analyze and print the data
How Real-Time PCR Works
Real-time PCR analysis detects specific nucleic acid amplification products as they accumulate in real-time. Real-time PCR uses a fluorescently labeled oligonucleotide probe, which eliminates the need for post-PCR processing. It is capable of screening genetic activity within hours using a minimal amount of sample material, and can detect a single molecule of DNA or RNA.
During polymerization, a reporter fluorescence dye and a quencher dye are attached to a TaqMan® probe. Negligible fluorescence from the reporter dye's emission is observed once both dyes are attached to the probe. Once PCR amplification begins, DNA polymerase cleaves the probe, and the reporter dye is released from the probe. The reporter dye, which is separated from the quencher dye during every amplification cycle, generates a sequence-specific fluorescent signal. The signal increases in real time as PCR cycles continue; the fluorescence intensity increases proportionally.
Diagram taken from TaqMan Gold RT-PCR kit protocol (PE Applied Biosystems)
RT-PCR addresses the problem of end-point analysis, which is commonly seen in traditional PCR assays. Too much amplification makes it impossible to quantitate the amount of starting nucleic acid material. RT-PCR monitors the product accumulation as the PCR amplification proceeds, allowing for higher sensitivity. Another advantage of real-time PCR is that the PCR tubes don't need to be opened after the amplification reaction is complete. This prevents contamination by PCR products and reduces the amount of "false positive" results.