Validation of Microarray results using Real Time PCR
Analysis of data from Real time experiments J.M.K. Mulema Department of Molecular and Cell Biology University of Cape Town 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 One step Vs Two step One step Two step cDNA synthesis to PCR amplification is Reverse transcription and PCR performed in a single tube. Minimizes experimental variation occur in separate tubes Allows several different real time because both enzymatic reactions PCR assays on dilutions of a single occur in a single tube cDNA. 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. 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 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 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 20l reaction. 1. At3g51660 (Macrophage migratory) 2. At4g30270 (MERI-5 protein) 3. At2g47190 (Myb family transcription) 4. At5g39610 (No Apical Meristem) 5. At5g06860 (PGIP1) 6. At4g24340 (Phosphorylase) 7. At5g07010 (Sulfotransferase) 8. At1g22400 (UDP-glucoronosyl) 9. At1g62300 (WRKY) Diluted the synthesized cDNA 1 in 10 10. At3g04720 (Hevein-like protein) Designed primers to amplify products ranging 12. At3g50480 (Broad spectrum) from 70-150 bp Amplification optimized with a conventional PCR before real time. 11. At3g28930 (avrRpt2-induced protein) 13. At2g24180 (CYP 450)
14. At3g04220 (Disease resistance protein) 15. At1g52200 (Expressed protein) 16. At2g39030 (GCN5-related acetyltransferase) 1. At4g10340 (Chlorophyll A-B) 17. At4g16260 (Glycosyl hydrolase) 2. At1g72610 (Germin-like protein) 18. At4g15610 (Integral membrane protein) 3. At1g12900 (GAPHD) 19. At4g33150 (lysine-ketoglutarate) 1. At5g25760 (Ubiquitin-conjugating enzyme) 4. At5g38430 (RUBSCO) 5. At1g20340 (Plastocyanin) 2. At5g06600 (Ubiquitin-specific protease) 3. At1g04820 (Tubulin alpha 2-alpha 4) Absolute/Relative quantification Used serial diluted standards of known concentration to generate a standard curve. Generating a standard curve 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 Standard curve is a linear relationship between the ct and the initial amount amounts of RNA or Pooled sample cDNA. This allows the determination of the Dilution 1 (10-1) concentration of unknowns based on
Dilution 2 (10-2) their ct values Dilution 3 (10-3) Assumes that all standards and samples have equal amplification efficiencies. Carry out runs in triplicates Source: Celtic Diagnostic Upregulated genes Gene 0hrs R1 12hrs R1 24hrs R1 0hrs R2 12hrs R2 24hrs R2 0hrs R3 12hrs R3 24hrs R3 At3g28930 11.756 81.5393 224.747 5.1133 33.1119 388.9217 8.6277 85.026 106.6237 At3g50480 64.3495 158.3122 179.2381 54.568 70.2542
17.71 16.5 18.5 18.27 17.93 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) Calculation of relative values (At2g24180) Replicate GOI HKG GOI/HKG Relative values 0 hrs Rep 1 13.7458 127.6356 0.107695659 1.000 12 hrs Rep 1 55.4771 183.0081 0.303140134 2.815 24 hrs Rep 1 309.4145 118.7417 2.60577792
12 hrs Rep 1 24 hrs Rep 1 0 hrs Rep 2 Time points At4g10340 At1g72610 At1g12900 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. 1.88E+02 Validated genes from Microarray 2.00E+02 1.80E+02 1.40E+02
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 x (1 + E) CT,X-CT,R Ro = XN x (1 + E) CT Calibration = XN,q x (1 + E) CT,q = XNcb x (1 + E) = (1 + E) CT CT,cb Amount of the target normalized to an endogenous reference and relative to the calibrator is given by Amount of target = 2-CT Assumption: The amplification efficiency of the target and reference are approximately equal - 0 0 0.5 1 1.5 2
0.033 1.2 1 Time points 0.8 0.6 0.4 0.2 0 0 hrs 0 hrs 0 hrs 12 hrs 12 hrs Amount of Target 12 hrs 24 hrs 24 hrs 24 hrs 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
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