Real-time PCR provides a more accurate way to quantify gene expression compared to older methods like Northern blotting. It works by measuring fluorescence at each PCR cycle, allowing quantification of mRNA levels based on the threshold cycle. Key advantages include a wide dynamic range, high precision, and not requiring post-PCR processing. Factors like primer and probe design, normalization, and data analysis influence reproducibility and accuracy of real-time PCR gene expression analysis.

1. Real Time PCR & its applications Dinesh Kumar M.Sc. Biotech(BHU), Ph. D. Biotech(BHU), PDF(USA) Senior Scientist (Animal Biotechnology) Genes & Genetic Resources Molecular Analysis Lab National Bureau of Animal Genetic Resources Karnal-132001 E-mail: dineshkumarbhu@gmail.com, +91-94161-11753(SMS only) 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

2. Why to measure gene expression? Chargoff's law two laws Traits vary Structural variants of gene Functional variants of gene Structural genomics to functional genomics

3. Why to quantify mRNA? Gene expression studies

4. Measurement of mRNA Cell- 100 mRNA of gene A Extract- Northern blotting-densiometry Extract-cDNA-template-PCR-threshold Accuracy in number? 40, 50, 60 molecules Accuracy loss Extraction Transfer/blotting Hubridisation/washing Signal/noise Reading/manual error 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

5. How to maximize the accuracy? Use of PCR Measure the threshold (Ct) Compare with house keeping genes. Relative gene expression. Straight line equation(range) PCR efficiency 100% How to do all these? 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

6. How to quantify mRNA? Northern blotting: In situ hybridization: RNAse protection assays: RT-PCR 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010 Gene XXXXx XXXX X GAPDH +/+ +/+ 1 2 3 kb 0.0 0.5 1.0 1.5 2.0 2.5 Thymus Spleen Lymph Kidney

7. How real time PCR machine works? 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

8. How the fluorescence monitoring is done in real time PCR machine? 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

9. Advantages of qPCR Wide dynamic range of quantification (7–8 log decades). High technical sensitivity (< 5 copies). High precision (< 2% CV of CT values). No post-PCR steps like running of gel or sequencing etc. Non-specific amplification can be detected by melt curve analysis of PCR products. Minimized risk of cross contamination. High throughput. Multiplex approach possible. Saves lot of time. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

10. Limitations of qPCR PCR product increases exponentially. Variation increases with cycle number. Increased variation after transformation to linear values. Overlap of emission spectra. Maximal four simultaneous reactions limit the bacterial diagnostics. Increased risk of false negative results where parasite genome changes very fast. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

11. Primers for RT Specific primers: decreases background priming: Random hexamers: maximizes the number of mRNA molecules thus often overestimate mRNA copy numbers. Oligo-dT primers: maximizes the number of mRNA molecules 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

12. Quantification of mRNA- in vitro 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

13. RNA-poor quality 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

14. Primer/Probe designing for qPCR Primer Express Molecular Beacon-3 Primer 3.0 OLIGO Tm calculator m-fold 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

15. Common qPCR Instruments ABI Prism 7700, 7000 (Perkin-Elmer–Applied Biosystems, Foster City, CA, USA) Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany) Biorad Instruments- i cycler Corbett Research-Rotor Gene Opticon-II, Chromo 4 (MJ Research) Stratagene (Mx4000) Techne-Cephid-Smartcycler 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

16. Basic chemistries used in qPCR 1. DNA-binding dyes SG (S) will not bind to ssDNA and the intensity of fluorescent signal is low b) SG (S) binds to dsDNA the fluorescent signal intensity (E) increases 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

17. Advantages of DNA-binding dyes This method obviates the need for target-specific fluorescent probes, but its specificity is determined entirely by its primers. Simple and fast. Melt curve analysis ensures specificity of amplified PCR products. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

18. Melt curve analysis 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

19. Limitations of DNA-binding dyes any double-stranded DNA generates fluorescence amplification of a longer product will generate more signal than a shorter one. amplification efficiencies different- inaccurate result. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

20. 2. Molecular beacons The hairpin structure causes the MB to fold when not hybridized, bringing quencher and fluorophore dyes in close proximity causing quenching of fluorescence. When hybridized, the fluorophore and quencher are separated resulting in increased fluorescence. Advantages of Molecular Beacons: specificity non-PCR amplification assays Limitations 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

21. Limitations of molecular beacons design of the hybridization probe. interfere with the hybridization 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

22. Hybridisation probes fluorescence resonance energy transfer ( FRET ) Resonance energy (E) transfer is low when the probes are not hybridised. Hybridisation of the probes brings the donor (D) and acceptor (A) fluorophores into close proximity resulting in increased resonance energy transfer. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

23. Advantages of Hybridization probes: increases specificity probes are not hydrolyzed, fluorescence is reversible :allows the generation of melting curves. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

24. Hydrolysis probes Energy (E) emitted by the donor (D) fluorophore is absorbed/quenched by the acceptor (A) fluorophore. The polymerase exonuclease activity separates the D fluorophore from the A fluorophore by hydrolysis resulting in an increase in fluorescent signal. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

25. Limitations of Hydrolysis probes reduces the processivity of the polymerases less efficient when mutation-specific RT-PCR 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

26. Amplicon design < 100 bp, no longer than 80 bp are found to be ideal as long as 400 bp minimum 63 bp . Primer and probe design 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

27. Primer & probe designing Primers 15-30 bp in length G/C content of 20-80% Avoid primer dimers. The Tm should be within 2°C Purify by gel electrophoresis or HPLC Optimize concentrations by performing matrix of 50nM, 300nM and 900nM fo the forward and reverse primers. This range allows the matching of the experimental Tm of the primers by +/- 2 ° Probes 20-30 bp in length G/C content of 20-80% Tm 7-10 ° higher than primers To maximize signal or reporter vary the probe concentration between 5-400nM 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

28. Data analysis in qPCR What is the Ct-value? 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

29. Amplification Plots Kumar SYBR Green, 12-18-2003, 10Hr 00Min.mxp.txt 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

30. Dissociation Curve Kumar SYBR Green, 12-18-2003, 10Hr 00Min.mxp.txt 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

31. Standard Curve Kumar SYBR Green, 12-18-2003, 10Hr 00Min.mxp.txt 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

32. What are the reaction efficiency, amplification and slope and how is it calculated? smaller the Ct value ( y intercept) in the regression equation, the greater the sensitivity of the RT-PCR reaction. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

33. Amplification Plots Kumar SYBR Green, 12-18-2003, 10Hr 00Min.mxp.txt 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

34. Factors affecting reproducibility of Ct When work with low copy number. Short amplicons have greater reproducibility. It is always better to use replicates for greater accuracy. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

35. Why and how to Normalize expression data? any variation in the amount of starting material a cellular RNA that serves as an internal reference against which other RNA values can be normalized. housekeeping genes (HKGs) GAPDH ,  -actin , rRNA H3 and cyclophilin, tubulin, micro-globulin etc 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

36. Absolute Quantification C (cRNA} µg/l N (molecules perµl) = __________________ X 182·5×10 13 K (fragment size /bp) Where, N= gene molecules per µl, C = concentration of the cRNA inµg/l, K= fragment size or bp, 182·5×10 13 Avagadro constant. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

37. Relative and Comparative Quantification relative to an active reference control (normalizer or house keeping genes) 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

38. Relative and Comparative Quantification Advantages No standard curve is required. More wells available for samples. Disadvantages More optimization and validation required If dynamic range of HKG and target genes are not of similar magnitude it needs correction factors. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

39. Mutation/allele detection A single base mismatch under the probe Decreases the Tm by as little as 3 0 C for G–T By 10 0 C for A–C mismatches. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

40. Applications Quantification Absolute Gene Expression Viral, Fungal, Bacterial Load Genomic Mitochondrial DNAs Quality Control Relative and Comparative Gene expression Microarray validation Detection Sequence Detection Allele discrimination SNP analysis Zygosity testing Zoonotic agents GMO screening Residual disease Splice variants Chromosomal translocations Methylation Fluorescence Detection Isothermal signal amplification Plate reader functionality 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

41. Multiplexing 4 channel machine Single PCR tube-data of 4 different genes Problems Advantages 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

42. Ten most common Real-Time qPCR Pitfalls Poor primer and probe design Poor quality RNA Not using “master mixes” Introducing cross contamination Not using a “-RT”control Using and inappropriate normalization control Melt curve analysis not done when using SYBR Green Baseline and threshold not set correctly Efficiency of the reaction is poor Using an inappropriate range for standard curve 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

43. Reaction set up 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

44. 01/06/10

45. Real time monitoring 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

46. Dinesh Kumar Scientist (Animal Biotechnology) Small Ruminant Molecular Genetics Lab DNA Fingerprinting Unit National Bureau of Animal Genetic Resources Karnal-132001 E-mail:dineshbhu@rediffmail.com, 094161-11753 Thanks ! I am thankful to Dr J.M.Reecy and his group for Making me happen to learn a b c of real time in real spirit! Thank you to every one here ! 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010