Abdelilah Soussi Gounni, Ph.D Associate Professor Manitoba Research Chair

1. Quantification of mRNA using real-time PCR Abdelilah Soussi Gounni, Ph.D Associate Professor Manitoba Research Chair Department of Immunology University of Manitoba

2. Objectives • Introduction to qPCR • Chemistries used in qPCR • Different platforms : pro and con • Primer design and probe design • RT: Methods of priming. • Sample preparation, RNA extraction. • Quantification strategies • QPCR application

3. Why Measure Gene Expression? • More abundant genes/transcripts are more important. • Each cell has a standard expression profile/signature. • Assumption that gene expression levels correspond to protein levels.

4. Why Measure Gene Expression? • Gene expression profiles represent a snapshot of cellular metabolism or activity at the molecular scale • Gene expression profiles represent the cumulative interactions of many intracellular events or phenomena

6. Real time PCR: Generalities • The real-time polymerase chain reaction uses fluorescent reporter dyes to combine DNA amplification and detection steps in a single tube format. • Increase in fluorescent signal is recorded during the assay. • Fluorescent signal is proportional to the amount of DNA synthesized during each amplification cycle.

7. Real time PCR: Generalities • Individual reactions are characterized by the cycle fraction at which fluorescence first rises above a defined  background fluorescence, a parameter known as the threshold cycle (Ct) or crossing point (Cp). • Consequently, the lower the Ct, the more abundant the initial target.  • The homogeneous format eliminates the need for post-amplification manipulation and significantly reduces hands-on time and the risk of contamination.

8. A hypothetical amplification plot • The amplification plot is the plot of fluorescence signal vs PCR cycle number. • The baseline is defined as the PCR cycles in which a signal is accumulating but is beneath the limits of detection of the instrument.

9. Real time PCR some jargons to start with… • The signal measured during these PCR cycles is used to plot the threshold. • The threshold is calculated as 10 times the standard deviation of the average signal of the baseline fluorescent signal. • A fluorescent signal that is detected above the threshold is considered a real signal that can be used to define the threshold cycle (Ct) for a sample. • The Ct is defined as the fractional PCR cycle number at which the fluorescent signal is greater than the minimal detection level.

12. Three main chemistries are used in Real time PCR 1- Intercalating dyes SYBR-Green: this dye fluoresce upon light excitation when bound to double stranded DNA. • Advantage Cost effective. Easily added to assays Amplification products can be verified by the use of melting curves. More sensitive than probe-based assays. • Disadvantage: Lack of specificity and fluorescence varies with amplicon length.

13. Three main chemistries are used in Real time PCR 1- Intercalating dyes SYBR-Green

14. Three main chemistries are used in Real time PCR 1- Intercalating dyes SYBR-Green

15. 2. Hybridisation-probe based methods: TaqMan or Molecular Beacons. • Most specific, as products are only detected if the probes hybridize to the appropriate amplification products. Many variations on this theme, with melt curve analysis possible for some chemistries (UBI). Main disadvantages are cost, complexity and occasional fragility of probe synthesis. There are potential problems associated with the fact that probe-based assays do not report primer dimers that can interfere with the efficiency of the amplification reaction.

16. 2-TaqMan Probes • Neccesites 2 primers and one probe • Oligonucleotides that contain a fluorescent dye, typically on the 5' base, and a quenching  dye, typically located on the 3' base. • When irradiated, the excited fluorescent dye transfers energy to the nearby quenching dye molecule rather than fluorescing, resulting in a non-fluorescent substrate. • TaqMan probes are  designed to hybridize to an internal region of a PCR product.

17. 2-TaqMan Probes • During PCR, when the polymerase replicates a  template on which a TaqMan probe is bound, the 5' exonuclease activity of the polymerase cleaves the probe. •  This separates the fluorescent and quenching dyes and FRET no longer occurs. • Fluorescence increases in each  cycle, proportional to the rate of probe cleavage. 

18. TaqMan probe

19. 3- A variation of Taqman Fluorophores attached to primers • Invitrogen's Lux or Promega's Plexor primers. • Advantage: Relatively inexpensive and amplification products can be verified by melting curves. • Specificity depends on the primers. • Usually company-specific design software needs to be used for optimal performance.

20. LUX™ (light Upon eXtension) • Detection technology uses one primer labeled with a single fluorophore and a corresponding unlabeled primer. • Both custom-synthesized according to the target of interest. Typically 20-30 bases in length. • Primers are designed with a fluorophore near the 3´end in a hairpin structure. • This configuration intrinsically renders fluorescence quenching capability, making a separate quenching moiety unnecessary. • Once the primer becomes incorporated into the double-stranded PCR product, the fluorophore is de- quenched.

21. LUX™ (light Upon eXtension) Primers Invitrogen Web site

22. Platforms or Cyclers Two very different principles for commercial thermocyclers: block-and air based. The block based cyclers: • They worked by heating and cooling a metal block in which the samples are placed. • The heat is usually moved by Peltier elements which are semiconductors which move heat when a current is applied to the cells. • If the polarity of the current is reversed, the direction of the heat transport is also reversed.

23. Heating Block: disadvantage • Heating blocks are relatively slow and changes typically the temperature 1 to a few degree celcius per second. • Heating blocks are difficult to heat uniformly across the plate.

24. Air cyclers • The temperature changes by means of heated air. • The samples are in 96-well plates, or in capillaries, the later conduct the heat very rapidly. • Traditionally, air cyclers can typically accommodate less samples than plate cyclers, but the time to complete a run is much shorter, due to the rapid heat conduction in the system. Not anymore: Roche new system. • The temperature gradient is typically 20 degrees C per second.

25. Designing primers Look at public data bases first: • Rt primer DB: htpp://medgen.ugent.be/rtprimer db • Primer bank htpp://pga.mgh.harvard.edu/primerbank/index. Html • Real-time primers set(http://www.realtimeprimers.org

26. Conti.. If you found your probe and primers: • Then input the sequences into blast http://www.ncbi.nlm.nih.org • Examine the sequences for possible errors, polymorphisms and avoid these regions for primer or probe design. • Avoid direct repeat in the target sequences: hybridization to alternative site in repetitive regions results in non productive binding of primers, a reduction in the efficiency of DNA amplification. • Sensitivity reduction of the assay

27. Conti... -When possible use a primer sequence in boundaries between two exons separated by along introns : no need for DNAase treatment due to genomic contamination. -Try to have a short amplicon as possible (60 to 150 bp) with GC content of 60 % or less to ensure efficient denaturation.

28. Isolation of RNA • RNA isolation based on 3 principals • Lysing of cells to release nucleic acids • Inactivation of RNases • Separation of RNA from DNA • Old School • Tissue Homogenization (Grinding on Dry Ice/LN2) • Hot Phenol/SDS or Guanidium thiocyanate • CsCl gradient centrifugation • New • Chaotropic Salt Isolation (Qiagen) • Trizol (Invitrogen) • Isolation of mRNA from total RNA (oligo dT) • Isolation of cytoplasmic RNA

29. RNA assessment • Many strategies are used • Spectrophotometer: widely used • Riobogreen • Agilent bioanalyser • Nanodrop • Biorad experion • No method leads to the same data!! • Extremely important: Use the same methods for measuring all samples • Do not compare data of samples analysed by different methods Butin SA et al 2005

30. RNA quality • Widely used approaches : • ratio A260/280; between: 1.9 to 2.1 ( 100 % purity) • Gel analysis of ribosomal RNA; intact 18S and 28S (doubling intensity on gel of 28S compared to 18S). • Agilent bio-analyzer/ BioRad experion microfluidic • Nanodrop • Free of contamination : • DNA • Proteins • Inhibitors: carry over chemicals from purification steps These contamination may depends on the tissue or cell from which RNA come from.

31. RNA quality • Best approach use 3’- 5’ assay using GAPDH as target gene. • Adopted by microarray users • Independent of ribosomal integrity • Accepted as conventional techniques applied to end point PCR assays Auer et al, 2003

32. Primers and amplicon for inhibitors assessment and 3’-5’assay

33. RNA quality assay

34. Effect of Primers concentration on QPCR Optimum: The lowest Ct and the highest normalized fluorescence Nolan T et al, 2006

35. cDNA priming • Oligo dT: • suppose that the RNA is intact. • non suitable if looking for: • Splice variants • Sequences long 3 UTR • Non poly genes

36. cDNA priming • Random hexamers: • not equal efficiencies of RT of all target genes. • Absence of linear correlation between input RNA and cDNA yield when a specific target is measured. • Gene specific primers: • Good choice if the RNA is not limited. • Useful for extremely rare mRNA.

37. cDNA Priming • 15nt Random primers: tend to be the choice taking in account recent studies comparing all methods. • This method has been shown to yield 80% of the template compared to 40% with random hexamers RT step tends to be the source of variability in PCR

38. Practical lab considerations

39. Prepare well all your samples ( start with a simple experiment first). • Be sure that you have all what you need. • Never use other people reagents. • Work in dedicated area for RT-QPCR. • Prepare all the controls.

40. Analysing and quantification of Real time PCR data • Three methods • Absolute quantification: use absolute standard curve. Measures the ACTUAL number of molecules of the target • Relative quantification: use relative standard curve or • ΔΔ CT method does not require standard curve. But you have to perform validating step experiment. The last 2 both quantify relative changes of the target gene compared to calibrator.

41. Functions of Standard Curves 1-Take efficiency into account. *In relative standard method, efficiencies of gene assays allowed to differ . As such, slopes of standard curves correct for efficiency. *With comparative Ct method, efficiencies of target and control genes must be approximately equal. Efficiency ? What we are talking about ?

42. Equation for the Theory of Quantitative PCR y = x(1+e)n x = starting quantity y = yield n = number of cycles e = efficiency

43. Functions of Standard Curves 1-Take efficiency into account. *In relative standard method, efficiencies of gene assays allowed to differ . As such, slopes of standard curves correct for efficiency. *With comparative Ct method, efficiencies of target and control genes must be approximately equal. 2- Convert Ct’s to quantity units 3- Test dynamic range

44. Acceptable Standard curve

45. Unacceptable standard curve

46. Ct : threshold cycle • The cycle at which the fluorescence exceeds a detection threshold, the Ct (threshold cycle) correlates to the number of target cDNA molecules present in the added cDNA. • Consequently, by comparison to a calibration curve, it is possible to quantify in absolute amounts the number of target molecules in added cDNA samples.

47. Relationship between standard curve and amplification

48. Comparative Ct method • The Ct values of different samples are used to calculate the relative abundance of template for each sample. • In this plot, the solid line crosses the threshold at PCR cycle number 18 whereas the dotted line crosses at 20. • By subtracting 18 from 20, there is a two-cycle difference between these two samples or a ΔCt of 2. • Due to the exponential nature of PCR the ΔCt is converted to a linear form by 2−(ΔCt) or fourfold difference. • This calculation is used when performing a relative quantitation analytical method.

50. Dissociation curves are useful for determining the presence of multiple species in the sample • Every PCR product melts at a characteristic temperature, its Tm (melting temperature). • A characteristic melting peak at the amplicon's Tm will usually distinguish it from amplification artifacts that melt at lower temperatures in broader peaks. • In the PCR, these are typically primer–dimer artifacts or non-specific amplicons. • For standard analysis, samples are first melted at 95 °C, and then equilibrated at 60 °C before being slowly re-heated (dissociated) back to 95 °C. The centre of each peak reflects Tm.