Analysis of data from Real time experiments

1. Analysis of data from Real time experiments J.M.K. Mulema Department of Molecular and Cell Biology University of Cape Town

2. Introduction • Quantifying RNA; Northern blotting, In situ hybridization, RNAse protection assays, Microarray, RT-PCR. • Real time PCR • Data collected throughout the PCR process as it occurs. • Amplification and detection combined into a single step. • Reactions characterized by the point in time where the target amplification is first detected. • Cycle threshold (Ct), the time at which fluorescent intensity is greater than background fluorescence. • Greater starting target DNA, faster significant increase in fluorescent, lower Ct • Requires much less RNA template

3. One step Vs Two step • Two step • Reverse transcription and PCR occur in separate tubes • Allows several different real time PCR assays on dilutions of a single cDNA. • Reactions from subsequent assays have the same amount of template to those assayed earlier. • Date from two step is quite reproducible. • Allow for increased DNA contamination. Design the target PCR product to span introns • One step • cDNA synthesis to PCR amplification is performed in a single tube. • Minimizes experimental variation because both enzymatic reactions occur in a single tube • Uses RNA starting template. Prone to rapid degradation if not handled well. • Not suitable in situations where the same sample is assayed on several occasions over a period of time. • Less sensitive than two step protocols.

4. Genome sequenced • A rapid life cycle • Prolific seed production • Cultivation in restricted space • Easily transformed • Botrytis cinerea (anamorph) • Botryotinia fuckeliana (teleomorph) • Ubiquitous • Chemical control • Host range Identify genes that play a role in resistance

5. At3g51660 (Macrophage migratory) • At4g30270 (MERI-5 protein) • At2g47190 (Myb family transcription) • At5g39610 (No Apical Meristem) • At5g06860 (PGIP1) • At4g24340 (Phosphorylase) • At5g07010 (Sulfotransferase) • At1g22400 (UDP-glucoronosyl) • At1g62300 (WRKY) • At3g04720 (Hevein-like protein) • At3g28930 (avrRpt2-induced protein) • At3g50480 (Broad spectrum) • At2g24180 (CYP 450) • At3g04220 (Disease resistance protein) • At1g52200 (Expressed protein) • At2g39030 (GCN5-related acetyltransferase) • At4g16260 (Glycosyl hydrolase) • At4g15610 (Integral membrane protein) • At4g33150 (lysine-ketoglutarate) • Arabidopsis leaves infected with B. cinerea • Extracted RNA from first three replicates (untreated, 12 hrs pi and 24 hrs pi) • Made overnight cDNA synthesis • Used Superscript III reverse transcriptase in a 20µl reaction. • Diluted the synthesized cDNA 1 in 10 • Designed primers to amplify products ranging from 70-150 bp • Amplification optimized with a conventional PCR before real time. • At4g10340 (Chlorophyll A-B) • At1g72610 (Germin-like protein) • At1g12900 (GAPHD) • At5g38430 (RUBSCO) • At1g20340 (Plastocyanin) • At5g25760 (Ubiquitin-conjugating enzyme) • At5g06600 (Ubiquitin-specific protease) • At1g04820 (Tubulin alpha 2-alpha 4) • At5g44200 (Nuclear cap-binding protein)

6. Rep 1 0 hrs 12 hrs pi 24 hrs pi Rep 2 0 hrs 12 hrs pi 24 hrs pi Rep 3 0 hrs 12 hrs pi 24 hrs pi Source: Celtic Diagnostic • Absolute/Relative quantification • Used serial diluted standards of known concentration to generate a standard curve. • Standard curve is a linear relationship between the ct and the initial amount amounts of RNA or cDNA. • This allows the determination of the concentration of unknowns based on their ct values • Assumes that all standards and samples have equal amplification efficiencies. • The concentrations of serial dilutions should encompass all samples and stay within the range that can be quantified Generating a standard curve Pooled sample Dilution 1 (10-1) Dilution 2 (10-2) Dilution 3 (10-3) Carry out runs in triplicates

9. Upregulated genes

10. Down regulated genes Reference genes (Relative Conc) Reference genes (Ct values)

11. Types of real-time quantification • Absolute quantification • Uses serially diluted standards of known concentration to generate a standard curve. • PCR standards • Fragment of double stranded DNA • Single stranded DNA • Complementary RNA bearing the target sequence • Relative quantification • Changes in gene expression are an external standard or calibrator • Two standard curve method • Comparative Ct (Delta Delta Ct) method • Pfaffl method – Relative expression software tool (REST)

12. Calculation of relative values (At2g24180)

14. Relative expression software tool (REST) • Purpose is to determine if there is a significant difference between samples and controls taking into account issues of reaction efficiency and reference gene normalization. • Randomization are used in Rest. • The alternate hypothesis P(H1) is based on the assumption that the difference between sample and control groups is due only to chance. • REST performs 50,000 random reallocations of samples and controls between the groups and counts the number of times the relative expression of the randomly assigned group is greater than the sample data • Concentration = efficiencyavg(Controls) – avg(Samples) • Expression = goiConcentration ÷ refConcentration • Expression = GEOMEAN(goiConcentration ÷ refConc1, goiConcentration ÷ refConc2, …) • Calculate a normalization factor equal to the geometric mean • REST performs its calculations based on CP and efficiency values determined by standard curve or kinetic techniques.

19. Xn = Xo x (1 + Ex)n XT = Xo x (1 + Ex)CT,X=KX RT = Ro x (1 + ER)CT,R=KR Dividing XT by RT gives the expression XT = Xo x (1 + Ex)CT,X=KX RT = Ro x (1 + ER)CT,R=KR Assuming equal amplification efficiencies EX =ER = E Xo Ro = XN,q x (1 + E) ΔCT,q = (1 + E) -ΔΔCT = XNcb x (1 + E) ΔCT,cb Delta Delta Ct method Calibration Amount of the target normalized to an endogenous reference and relative to the calibrator is given by Amount of target = 2-ΔΔCT Assumption: The amplification efficiency of the target and reference are approximately equal x (1 + E) CT,X-CT,R = XN x (1 + E) ΔCT

23. Conclusion • Real Time PCR is a powerful technique that gives quantitative answers difficult to obtain with end point PCR, however, all steps need to be controlled from sampling to PCR including manipulations like extraction and reverse transcription