RT-PCR Protocol

RT-PCR (reverse transcription-polymerase chain reaction) is a variant of the polymerase chain reaction (PCR) which are now widely used. Traditionally RT-PCR involves two steps: the RT reaction and PCR amplification. RNA is first reverse transcribed into cDNA using a reverse transcriptase as described here, the resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes. RT-PCR can be used to quantify mRNA levels from much smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell.

Experiment process

(1) Primer design

Design and synthesize the primers of the target gene. In general, primers should be highly specific for the target sequences, able to form stable duplexes with their target sequences, and free of secondary structure. Primers around 20~25 nucleotides in length should be synthesized. The GC content of these primers is designed as similar as possible. The total length of amplification is about 80~200bp.

(2) RNA extraction

Total RNA is extracted from cells or tissues of the plants or animals taking a standard extraction protocol with Trizol, dissolved in DEPC-treated deionized water and quantified with spectrophotometer. For RNAs with polyadenylated tails, enrich them with mRNA Purification Kit.

(3) Reverse transcription(RNA→cDNA)

RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase. Subsequently, the newly synthesized cDNA is amplified using traditional PCR. The total RNA is used as a template and Oligo (dT) or random primers and reverse transcriptase are used to reverse transcription into cDNA.

(4) Real-time PCR

Real-time PCR (qPCR) is the method of choice for quantification of gene expression. It is the preferred method of obtaining results from array analysis and gene expressions on a global scale. Currently, there are four different fluorescent DNA probes available for the real-time PCR products detection: SYBR Green, TaqMan, Molecular Beacons, and Scorpions. All of these probes allow the detection of PCR products by generating a fluorescent signal. While the SYBR Green dye emits its fluorescent signal simply by binding to the double-stranded DNA in solution, the TaqMan probes, Molecular Beacons and Scorpions generation of fluorescence depend on Förster Resonance Energy Transfer (FRET) coupling of the dye molecule and a quencher moiety to the oligonucleotide substrates.

(5) Result analysis

In the initial few cycles of the qPCR amplification reaction, the fluorescence signal changes little and approaches a straight line, which is the baseline. The fluorescence signals of the original 15 cycles is the background signal of fluorescence. The fluorescence domain values are 10 times the standard deviation of 3-15 circular fluorescent signal, and threshold set in the exponential phase of the PCR amplification. The CT value indicates the number of cycles experienced by each PCR reaction tube when the fluorescence signal reaches the set domain value.The more number the template onset copy has, and less the Ct value will beand vice versa. Generating consistent amplification across a wide dynamic range is fundamental to qPCR methodology, and use the △△t method for gene expression analysis.

The factors affecting Real-time PCR results

1. Reaction liquid composition: The fluorescence emission is affected by environmental factors, such as pH and concentration of metallic ion solution of reaction liquid.
2. ROX reference fluorescence: Lowering the concentration of ROX will increase the standard deviation of the CT value, and there are lower credibility of the small differences between the target templates.
3. Reaction efficiency of PCR: Different PCR amplification efficiencies may produce standard curves with different slopes. The efficiency of PCR amplification depends on the experiment, performance of reaction mixed liquid and sample quality.
   In order to improve the efficiency of PCR amplification, more than five different magnitude gradients should be performed. At least three repetitions should be done to improve precision. And the number of repetitions of low concentration samples should be increased to increase sensitivity. Through these methods, we can improve the accuracy of RT-PCR.