1. RealTime-PCR

3. Results

4. What’s Wrong With Agarose Gels?
Low sensitivity
Non-automated
End point analysis

5. Working of QPCR?
Real Time PCR is a technique in which probes bind to specific target regions of amplicons (GOI) to produce fluorescence during PCR.
The fluorescence, measured in Real Time, is detected in a PCR cycler with an inbuilt filter flurometer.
In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length (usually 20–30 bases long) which can be radioactively labeled. It can then be used in DNA or RNA samples to detect the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe.

6. Probe types & Design

7. dsDNA BindingDye
SYBR Green I
TaqMan Probe

8. Sybr Green PCR Assay Stronger signal Higher selectivity for dsDNA
Lesser sequence dependent
Higher stability
Binds to
Non specific PCR product
Primer dimer

9. TaqMan Probe

10. TaqMan Probe
When intact, the fluorescence of the reporter is quenched due to its proximity to the quencher
Probe hybridizes to the target
dsDNA-specific 5'—>3' exonuclease activity of Taq cleaves off the reporter
Reporter is separated from the quencher.
Fluorescent signal
Signal is proportional to the amount of amplified product in the sample

11. TaqMan Probe Advantages Disadvantages Highly fluorogenic
Easy PCR setup
Sequence-specific detection
Disadvantages
Expensive
Probe design and positioning challenging
Similar conditions for primers and probes

12. Real-Time PCR Terminology
Amplification plot is the plot of fluorescence signal versus cycle number.
Initial cycles of PCR, there is little change in fluorescence signal. This defines the baseline of amplification plot.
An increase in fluorescence above the baseline indicates detection of accumulated PCR product.
The parameter CT(Threshold cycle) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold.

13. Ct Values: 25 23 28

14. Reverse Transcription (RT) PCR
RT-PCR : a highly sensitive technique for the detection and quantitation of mRNA (messenger RNA). The technique consists of two parts: The synthesis of cDNA (complementary DNA) from RNA by reverse transcription (RT) and. The amplification of a specific cDNA by the polymerase chain reaction (PCR).

17. Relative Quantification
Housekeeping gene: Abundantly and constantly expressed gene. Expression level of these genes remains constant. eg 18 S rRNA, GAPDH, β Actin
Normalization: To accurately quantify gene expression, the measured amount of RNA from the gene of interest is divided by the amount of RNA from a housekeeping gene measured in the same sample to normalize for possible variation in the amount and quality of RNA between different samples.

18. REALTIME-PCR APPLICATIONS

19. Application in Molecular Diagnostics
Clinical microbiology and Food microbiology
Gene expression
viral quantitation
Single Nucleotide Polymorphism (SNP) analysis
Cancer
Analysis of cellular immune response in peripheral blood
Chromosome aberrations