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.

1. RT-PCR ProtocolRT-PCR Protocol
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2. Introduction
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

3. Experiment process

4. (1) Primer design
We 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.

5. (2) RNA extraction
Total RNA is extracted from cells or tissues of the plants or animals
according to standard trizol protocol, dissolved in DEPC-treated deionized water
and quantified with spectrophotometer. For RNAs with polyadenylated tails,
enrich with a mRNA Purification Kit.

6. (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.

7. (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 analyses 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.

8. (5) Result analysis
In the initial few cycles of the PCR 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 be
and vice versa. Generating consistent amplification across a wide dynamic range
is fundamental to qPCR methodology .

9. The factors affecting RT-PCR results
• Reaction liquid composition: The fluorescence emission is affected by
environmental factors, such as pH and concentration of metallic ion solution
of reaction liquid.
• 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.
• 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.

10. Solution
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.

11. • Related Products and Services in Creative Biogene:
• RT-PCR Kits
• cDNA Synthesis Kits
• Reverse Transcriptase
• Real Time PCR

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