REAL TIME PCR
Real Time in RT PCR denote it can monitor the progress of the amplification when the process is
going on.It is the most sensitive technique for mRNA detection and quantitation. 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.
Working procedure of RT PCR
Preformulated PCR master mixes containing buffer, DNA polymerase, and dNTPs are
commercially available from several vendors and can be used in combination with TaqMan probes.
As with SYBR® Green I assays, an optimized TaqMan assay should be sensitive and specific, and
should exhibit good amplification efficiency over a broad dynamic range
DNA Polymerases
During the elongation phase of real-time qPCR, a thermostable DNA polymerase, such as Taq, is used
to synthesize the new strand of DNA that is complementary to the template DNA strand. Taq is a
common thermostable DNA polymerase, native to the thermophillic bacterium Thermus aquaticus,
that has an optimum temperature for activity between ~70 and 80°C.
1. Denaturation: High temperature incubation is used to "melt” double- stranded DNA into single
strands and loosen secondary structure in single-stranded DNA. The highest temperature that the
DNA polymerase can withstand is typically used (usually 95°C). The denaturation time can be
increased if template GC content is high.
2. Annealing: During annealing, complementary sequences have an opportunity to hybridize, so an
appropriate temperature is used that is based on the calculated melting temperature (Tm) of the
primers(5°C below the Tm of the primer).
3. Extension: At 70-72°C, the activity of the DNA polymerase is optimal, and primer extension
occurs at rates of up to 100 bases per second.
When an amplicon in real-time PCR is small, this step is often combined with the annealing step
using 60°C as the temperature.
4. Detection: The detection is based on fluorescence technology.
The specimen is first kept in proper well and subjected to thermal cycle like normal PCR but at this
machine it is subjected to tungsten or halogen source that lead to fluoresce the marker added to the
sample and the signal is amplified with the amplification of copy number of sample DNA. The
emitted signal is detected by an detector and sent to computer after conversion into digital signal that
is displayed on screen. The signal can be detected when it comes up the threshold level(lower
detection level of detector), this cause elimination of background noise.
There are many different markers used as the marker of Real Time PCR. There are mainly two types
of marker are used for this purpose.
1. Taqman Probe: This is a hydrolysis probe, it bear a reporter dye , often fluorescein(FAM) at its 5’
end and a quencher tetramethylrhodamine (TAMRA), attached to the 3’ end of a oligonucleotide .In
normal condition the probe remain coiled on itself bringing the fluorescence dye near the quencher
causing quenching of fluorescent signal of the dye.
2. SYBR Green: This is a dye that provide prominent fluorescent signal when bind at the minor
groove of DNA Nonspecifically. Other fluorescent dyes like Ethidium Bromide or Acridine Orange
can also be Used but SYBR Green is better used for its higher signal intensity.
SYBR Green (Blinded at the minor Groove) Compare of fluorescent signal obtained from different
DNA binding dyes
SYBR Green is better then the Taqman Probe as it can provide the information about each cycle as
well as about the melting temperature that is not obtained by Taqman probe
Quantitation of Results
Standard Curve Method
In this method, a standard curve is first constructed from an RNA of known concentration. This curve
is then used as a reference standard for extrapolating quantitative information for mRNA targets of
unknown concentrations. Though RNA standards can be used, their stability can be a source of
variability in the final analyses. In addition, using RNA standards would involve the construction of
cDNA plasmids that have to be in vitro transcribed into the RNA standards and accurately
quantitated, a time-consuming process. However, the use of absolutely quantitated RNA standards
will help generate absolute copy number data.
Comparative Ct Method
Involves comparing Ct values of the sample with a control or caribilator such as non-treated sample.
The Ct values of both the calibrator and the samples are normalized to an endogenous housekeeping
gene. This give ∆Ct value of control and the sample
Comparative Ct method is also known as 2-∆∆Ct method where ∆∆Ct = ∆Ct,sample -∆Ct, reference
Fold Change = Efficiency-∆∆ Ct or 2-∆∆Ct (which gives relative gene expression)
Applications:
1. Gene Expression Analysis. 2. microRNA & Noncoding RNA Analysis. 3.Genetic Variation
Analysis. 4. Protein Analysis. 5. Mutation Detection. 6. Biomarker Analysis.
