1. Dr. Chaim Wachtel Introduction to PCR and qPCR Part II: PCR!!

2. qPCR technical workflow Sampling DNA Extraction RNA Extraction DNase treatment Reverse Transcription qPCR Data Analysis

3. Primer design

4. Primer design – key to successful PCR Good primer design saves time and money Advanced applications require even more stringent primer design –Multiplex –Low abundance

5. Good primer (pair) properties Primers should have 18-24 bases 40-60% G/C Balanced distribution of G/C and A/T bases T m that allows annealing at 55-65°C No internal secondary structures (hair-pins) Primer pairs should have Similar melting temperatures, T m, within 2-3 °C No significant complementarity (> 2-3 bp) –particularly not in the 3’-ends

6. ´3 Cycling... Sense Antisense Sense Antisense Sense cAntisense Antisense The primer dimer (PD) problem Primers that interact are amplified by PCR. PD formation competes with the designed PCR and can compromise the reaction efficiency.

7. Solution to the PD problem Reduce the formation of PDs by –Good primer design (avoid 3’ complementarity) –Minimal annealing time –Good laboratory practice –HotStart –TouchDown Reduce the signal from PDs by –Measuring fluorescence above the T m of the PDs –Use sequence-specific probe

8. Considerations Avoid targets with secondary structure Avoid pseudogenes Avoid genomic contamination by designing primers to span intron-exon-junctions exons introns PCR primers

9. Links for designing primers http://www.tataa.com/ http://www.ncbi.nlm.nih.gov/BLAST/ www.premierbiosoft.com/netprimer/netprlau nch/netprlaunch.htmlwww.premierbiosoft.com/netprimer/netprlau nch/netprlaunch.html www.ensembl.org http://www-genome.wi.mit.edu/cgi- bin/primer/primer3_www.cgihttp://www-genome.wi.mit.edu/cgi- bin/primer/primer3_www.cgi http://www.bioinfo.rpi.edu/applications/mfo ld/dna/form1.cgihttp://www.bioinfo.rpi.edu/applications/mfo ld/dna/form1.cgihttp://www.bioinfo.rpi.edu/applications/mfo ld/dna/form1.cgihttp://www.bioinfo.rpi.edu/applications/mfo ld/dna/form1.cgi Primer Design- Beacon Designer/AlelleID Primer express 3 (AB)Primer express 3 (AB)

10. Primer Express Located on Software 1 Easy to use Not fool-proof, but none of them are…..

15. Primer design-work flow Find sequence Find sequence Design Primers Design Primers Primer3 or similar software Check Primers Check Primers for desired parameters - Tm - amplicon size - secondary structure - complementarity - specificity Netprimer, BLAST and similar software Satis-factory? No Run PCR Run PCR Yes …and gel electrophoresis to check specificity and functionality NCBI or Ensembl

16. TaqMan Probe Design Amplicon size 70-150 bp Tm of probe 68-70 °C G/C content 30-70% No G at the 5´end Avoid runs of identical nucleotides Avoid secondary structure Avoid complementarity with primers HPLC purification

17. Popular dyes and quenchers FAM JOE HEX TET VIC ROX Cy DABCYL TAMRA Black Hole Quenchers

18. RT-PCR Housekeeping genes –What are they –How do you choose Standard curve Primer Dimer Melt curve Optimization Test samples Reference

19. Workflow – preliminary data analysis Examine amplification curvesCheck/adjust baselineCheck/adjust thresholdCheck your controls (NTC/positive controls/No-RT)Check your samplesPerform experiment specific analysis

20. Baseline settings Baseline - is the initial cycles in PCR where there is little change in fluorecence signal, usually cycle ~3- 15 Set the baseline Fixed number of cycles Adaptive baseline Control baselines in the linear scale (y-axis)

21. Raw data

22. Baseline adjustment

23. The different phases Exponential growth phasePlateau phase Part of exponential growth phase where signal > background (noise) Samples must be compared in the exponential phase

24. Setting threshold Purpose: Find a level of fluorescence where samples can be compared The theoretical cycle where a sample intersect the threshold is called C t Linear scale Logarithmic scale Threshold level C t values Log

25. Setting threshold C t (threshold cycle): Threshold cycle reflects the cycle number at which the fluorescence generated within a reaction crosses the threshold. It is inversely correlated to the logarithm of the initial copy number

26. Setting threshold Several methods available for threshold setting –Standard deviation of the noise for the first few cycles –Second derivative maximum (SDM) –Best fit of standard curve (highest r 2 ) –Manual setting A two-fold difference in copy number should have one Ct difference no matter where the threshold is set within the exponential phase

27. Dilution series and standard curves Used to control the quality of your assays Absolute quantification –Standards = Diluted templates of known concentration –Standard curve = C t of each standard sample is plotted against the known concentration –Used to determine concentrations of unknown samples –Absolute quantification is dependent on the quality of the standard curve

28. Standard curve Comment: Always cover the whole range of sample concentrations.

29. Interpretation of the standard curve Linear regression Y = ax + b a = slope that gives efficiency of PCR from 10 –1/a = 1 + efficiency b = # of cycles for detecting one molecule

30. 0 5 10 15 20 25 30 1.E+031.E+041.E+051.E+061.E+071.E+081.E+09 Concentration (log scale)

31. Relative quantification Often there is no good standard available Compare amount with reference Reference genes Genomic DNA Spike Ribosomal RNA Example –Expression of target gene is 10% of the expr. of housekeeping gene. –Same gene in other tissue, expression is 100%.

32. Comparing treatments Finding the change in expression of the target gene in the sample compared to the control (no treatment) as a ratio, using a reference gene

33. MIQUE Nomenclature MIQUE - Minimum Information for Publication of Quantitative Real-Time PCR experiments Suggested nomenclature Reference genes not housekeeping genes Quantification not quantitation Hydrolysis probes not TaqMan probes Quantification cycle Cq replaces Ct, Cp, TOP

34. Melting curve analysis Melting curves are obtained by measuring the fluorescence while increasing temperature Use a dye binding to double stranded DNA 7095 Temp 8090

35. Melting curve analysis Confirms formation of the expected product (each dsDNA has its characteristic melting temp T m ) Distinguishes between specific PCR products and non-specific products (e.g. primer-dimers) High resolution melt – mutation and methylation analysis

36. Melting curve Melting temperature T m is characteristic of the %GC, length and sequence.  The product can be identified from the T m. T m = 90 °C T m = 81.5 °C derivative 1 st

37. 4-steps PCR 4-steps PCR can be used to eliminate primer-dimer signals 40 cycles

38. Example – 4 steps PCR

41. 100% efficiency75% efficiency

42. 100% efficiency90% efficiency

43. 80% efficiency50% efficiency

44. 100%90%80%75%50% GMNv13.3053.8234.4564.8267.47 VN13.1863.4513.7753.9244.991 Ratio1.041.111.181.231.5 Reference primer efficiency

45. RT-PCR –testing samples ALWAYS perform melt curve ALWAYS run negative controls –No RT –No template Always Always Always run standard curve Triplicate of each sample!!

46. Requirements for RT-PCR Experiment Always perform standard curve All samples in triplicate NTC control No RT control Prepare mix without cDNA; add this to each tube separately Divide plate by gene and not sample Do not need reference gene on every plate Melt Curve Check RNA- otherwise don’t bother with experiment Do not rely on only 1 reference gene- check more than one per project Every project is different! Don’t be afraid to ask me questions, especially BEFORE starting the project.

47. Digital PCR From Relative quantity to absolute quantity

49. Commercially available machines Fluidigm QuantaSoft (Life Technologies)

50. Rain Dance Bio Rad QX100