Assay Design Considerations, Optimization and Validation Ray Meng, Ph.D. International Field Applications Specialist Gene Expression Division Bio-Rad Laboratories, Inc.
Assay Design Considerations
Experiment Considerations AMPLIFICATION Sample Preparation Quality and homogeneity of sample Sample Extraction Source of inhibitors Template preparation RNA extraction, quality, quantification Reverse transcription Strategy Experiment setup Technical replicates Biological replicates Optimization of primers and probes
Sample Preparation AMPLIFICATION Preparation Considerations RNA or DNA Source Homogeneity Prep time to extraction Sample degradation
Sample Extraction AMPLIFICATION DNA / RNA Aquapure TM Genomic DNA Isolation Kit Aurum TM Total RNA Kits Contaminants Starches Lipids Metals Extraction contaminants Phenol chloroform salts Sample degradation
Template DNA Preparation AMPLIFICATION Genomic DNA Cut with restriction enzyme that does not cut within amplicon Boil DNA for 10 min and then onto ice Plasmid DNA If it doesn t work, linearize plasmid with restriction enzyme that does not cut within amplicon cdna Treat RNA with RNase-free DNase prior to reverse transcription Use enzyme that has RNaseH activity to digest away RNA from RNA:DNA hybrid after making cdna.
Template Preparation AMPLIFICATION Extract and analyze RNA Careful quantification is necessary RiboGreen Assay - Quantification NanoDrop Quantification and purity Experion - Quality and quantification
Experion Analysis of RNA AMPLIFICATION Experiment: Evaluate sirna-mediated gene silencing Prevent faulty conclusions GAPDH sirna Scrambled sirna control A B C T 6.8 C T 4.3
Reverse Transcription Reproducible Data RNA Reality Ideal? cdna Not Reproducible
Reverse transcription Experiment: Testing Results Across a Range of cdna Input Concentrations iscript qrt-pcr Standard Curve Comparison: cdna serial dilution vs. total RNA serial dilution β-actin T 40 35 30 cdna Standard Total RNA Standard 25 20 y e 15 10 cdna total Slope -3.394-3.382 Corr. Coef. 0.999 0.999 Intercept 38.91 38.09 PCR efficiency 97.1% 97.6% RNA isolated from HeLa cells d u 5 1 2 3 4 5 6 7 8 9 Log Starting Quantity (femtograms of input RNA) Note: 1/10th of cdna reaction used for PCR
iscript based reagents AMPLIFICATION Reverse Transcription iscript TM cdna Synthesis Kit iscript Select cdna Synthesis Kit One-Step qrt-pcr iscript One-Step RT-PCR Kit with SYBR Green iscript One-Step RT-PCR Kit for Probes
Experiment setup Replicates Need for both technical replicates and Biological replicates. Number of replicates will depend on level of differences that are being presented. Lower expression genes tend to require more replicates to establish statistical validity of small differences. Biological Technical
Optimization of primers and probes AMPLIFICATION Hallmarks of an optimized real-time PCR assay: One specific product Good PCR efficiency Good intra- and inter-experimental reproducibility Sensitivity over a broad dynamic range Each hallmark can be tested experimentally. Spending more time on assay design means less time to achieve validated results.
Optimization of primers and probes Reaction Efficiency Reaction efficiency is 100% if product doubles at every cycle. Efficiency should be 100 +/- 10% Measure efficiency using a serial dilution of template Reactions designated as standards If template quantity is unknown, use 1.0, 0.1, 0.01, etc. Efficiency calculated based on standard curve slope
Optimization of primers and probes Efficiency (η) = [10 (-1/slope) ] - 1
Optimization of primers and probes AMPLIFICATION SYBR Green Validation Use a serial dilution of template to test primers across a broad dynamic range. Include representative unknown samples. Evaluate specificity, efficiency, reproducibility and dynamic range.
Optimization of primers and probes AMPLIFICATION Limit secondary structure 50 to 60% overall GC content Limit stretches of G or C s longer than 3 bases No Gs on the 5 end Place C s and G s on ends of primers, but no more than 2 in the last 5 bases on 3 end
Optimization of primers and probes AMPLIFICATION Perform BLAST searches on primers (and probe) and the target sequence. If starting with SYBR Green, design assay with the potential to use probes and to multiplex later. Test multiple primer combinations Find the primer pair with no primer-dimers and the best reaction efficiency
Assay Design
Amplicon Design AMPLIFICATION Length of 75 to 200 bp Limited secondary structure Model secondary structure using mfold, elaborate on salt and temp. http://bioinfo.math.rpi.edu/mfold/applications Avoid primer locations at stem loop structures
Probe Based Assays AMPLIFICATION Design primers first, test the reaction with SYBR Green, and then design the probe. For probe assays the fluorescence should be target specific, but the assay does not monitor PCR specificity. Amplicon size of 70-150bp Consider reporter fluorophore(s) for multiplexing.
TaqMan Design AMPLIFICATION Probe should have a Tm ~10oC higher than primers Tm of probe 68-70C G/C content 30-70% No G at 5 end Avoid identical nucleotide runs Avoid secondary structures Avoid dimerization with primers Select strand that gives more C than G bases
Primer Design Target Sequence 2 nd Structure Analysis Think Small Watch out for primer dimers NCBI HomePage Stacking No-Web Next Test in Real life conditions
Primer Design Free resource Blast sequence Stack sequence
Primer Design http://www.ncbi.nlm.nih. gov/blast/blast.cgi
Primer Design Target Sequence 2 nd Structure Analysis Think Small Watch out for primer dimers Mfold Dr. Zuker No-Web Next Test in Real life conditions
Amplicon Secondary Structures http://bioinfo.math.rpi.edu/mfold/applications Bad location for primers Good location for primers
Primer Design Reverse primer A Reverse Primer B η = 66.3 % Forward Primer Reverse Primer 1 110 A Reverse primer B η = 95.8 %
Primer Design Select target sequence: 1261 gatcgcaggg aagatggacc tgaagtcttc cagcaaactc aagaacgggc tcaccttccg 1321 caaggaagac atgcttcagc ggcagctcca cctggagggc atgctatgct ggaagaccac 1381 atcagggcgc ttgaaagata tcctggctat cctgctgacc gacgtacttt tgctgctaca 1441 agaaaaagat cagaaatacg tctttgcttc tgtggactca aagccacccg tcatctcgtt 1501 acaaaagctc atcgtgaggg aagtggccaa cgaggagaaa gcgatgtttc tgatcagcgc 1561 ctccttgcaa gggccggaga tgtatgaaat ctacacgagc tccaaagagg acaggaacgc 1621 ctggatggcc cacatccaaa gggctgtgga gagctgccct gacgaggagg aggggccctt 1681 cagcctgccc gaagaggaaa ggaaggtggt cgaggcccgc gccacgagac tccgggactt 1741 tcaagagcgg ttgagcatga aagaccagct gatcgcacag agcctcctag agaaacagca 1801 gatctacctg gagatggccg agatgggcgg cctcgaagac ctgccccagc cccgaggcct 1861 attccgtgga ggggacccat ccgagaccct gcagggggag ctaattctca agtcggccat Homo sapiens rho/rac guanine nucleotide exchange factor (GEF) 18 (ARHGEF18), mrna
Primer Design Dr. Michael Zuker s mfold http://www.bioinfo.rpi.edu/ applications/mfold/old/dna/ 55 o C
Primer Design Dr. Michael Zuker s mfold http://www.bioinfo.rpi.edu/ applications/mfold/old/dna/ 60 o C
Primer Design Dr. Michael Zuker s mfold http://www.bioinfo.rpi.edu/ applications/mfold/old/dna/ 65 o C
Primer Design Target Sequence 2 nd Structure Analysis Think Small Watch out for primer dimers Test in Real life conditions
Primer Design Remember: Keep things as simple and easy as possible
Primer Design Target Sequence 2 nd Structure Analysis Think Small Watch out for primer dimers Primer 3 Beacon Designer No-Web Next Test in Real life conditions
Primer Design Designs Primers Designs Internal Oligos Provides multiple outputs Free Web software provided by Steve Rozen and Whitehead Institute for Biomedical Research. http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
Primer Design Avoid Primer Dimers!! http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_help.cgi#primer_self_end
Primer Design Target Sequence 2 nd Structure Analysis Think Small Watch out for primer dimers Test in Real life conditions
Non validated primers IL-1b plasmid with SYBR detection 5-fold dilution series: 10,000 to 16 copies No resolution below 2000 copies r = 0.957 η = 153%
Non validated primers 10,000 copies 2,000 copies 400 copies No Template
Non validated primers Same primers with a specific probe Poor resolution Poor replicates η = 71%
Non validated primers After primer re-design to eliminate primer-dimers r = 0.999 η = 91.3%
Beacon Designer
RTPrimerDB
Assay Optimization and Validation
Fast Assay optimization dynamic thermal gradient
Fast Assay optimization Gradient Master Mix 16 wells + 2 (extras) @ 20 ul 360 ul total 180 ul 2X iq SYBR Supermix ul forward primer (200nM final) ul reverse primer (200nM final) ul DNA or cdna ul H 2 0 --------- 360 ul total Vortex!
Fast Assay optimization dynamic thermal gradient 10 o C Above 6 o C Below
Fast Assay optimization Melt curve
Fast Assay optimization Amplification } Real annealing range
Proper annealing conditions translates into better uniformity
Example of 12 replicates
Example of 12 replicates
Example of 12 replicates
Example of 12 replicates
Example of 12 replicates
Example of 12 replicates
Example of 12 replicates
Example of 12 replicates
Example of 12 replicates
Validation
Validation 1/3 1/3 1/3 1/3 Blank 10.0 ng/2ul 1.11 ng/2ul 0.12 ng/2ul 3.33 ng/2ul 0.37 ng/2ul
Validation
Validation
Validation
Validation
Validation
Validation
Validation Control Sample A Sample B T
Validation
Validation Test reaction product 2 kb β Actin ODC AZI OAZ 200 bp 100 bp 50 bp
In a Hurry?
In a hurry? If you are in a hurry to develop an assay design two or more primer sets and use the pair that gives the best results. Use low levels of template Earliest Ct Reproducible technical replicates Remember that being in a hurry is no excuse for not optimizing and validating your assay!
In a hurry? NF2 NF1 Actin NR1 NR2 NF1 NR1 NF2 NR2 NF1 NR2 NF2 NR1
Selecting the best primer set NF1 NR1 NF2 NR2 NF1 NR2 NF2 NR1
Good Primers AMPLIFICATION Specificity Melting curve analysis and gel analysis PCR Efficiency Slope of standard curve Reproducibility Standard deviations between replicates Sensitivity and dynamic range Experimental validation
Thank You! Questions?