assay design considerations, optimization and...

Assay Design Considerations,

Optimization and Validation

Ray Meng, Ph.D.International Field Applications Specialist

Gene Expression DivisionBio-Rad Laboratories, Inc.

Assay Design Considerations

AMPLIFICATION

www.bio-rad.com/genomics/pcrsupport

• 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

Experiment Considerations

AMPLIFICATION


Sample Preparation

• Preparation Considerations

– RNA or DNA

– Source

– Homogeneity

– Prep time to extraction

– Sample degradation

AMPLIFICATION


Sample Extraction

• DNA / RNA

– AquapureTM Genomic DNA Isolation Kit

– AurumTM Total RNA Kits

• Contaminants

– Starches

– Lipids

– Metals

• Extraction contaminants

– Phenol chloroform

– salts

• Sample degradation

AMPLIFICATION


Template DNA Preparation

• 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.

AMPLIFICATION


• Extract and analyze RNA

• Careful quantification is necessary

– RiboGreen Assay - Quantification

– NanoDrop – Quantification and purity

– Experion™ - Quality and quantification

Template Preparation

AMPLIFICATION


B

��CT 6.8

��CT 4.3

Sc

ram

ble

d s

iRN

A c

on

tro

l

GAPDH siRNA

A

Experion™ Analysis of RNA

• Experiment: Evaluate siRNA-mediated

gene silencing

• Prevent faulty conclusions

AMPLIFICATION


Reverse Transcription

IdealReality ?

RNA cDNA

Reproducible Data Not Reproducible

AMPLIFICATION


β-actin

iScript qRT-PCR Standard Curve Comparison:

cDNA serial dilution vs. total RNA serial dilution

Log Starting Quantity (femtograms of input RNA)

Note: 1/10th of cDNA reaction used for PCR

1 2 3 4 5 6 7 8 9

Cycle

old Nu

T

5

10

15

20

25

30

35

40

cDNA Standard Curve Total RNA Standard Curve

cDNA

total RNA Slope

-3.394

-3.382

Corr. Coef. 0.999

0.999

Intercept 38.91

38.09PCR efficiency 97.1% 97.6%

Reverse transcription

RNA isolated from HeLa cells

• Experiment: Testing Results Across a Range of cDNA Input Concentrations

AMPLIFICATION


iScript based reagents

Reverse Transcription

• iScriptTM 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

AMPLIFICATION


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.

TechnicalBiological

AMPLIFICATION


Optimization of primers and probes

• 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.

AMPLIFICATION



• 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

Reaction Efficiency

AMPLIFICATION



• Efficiency (ηηηη) = [10(-1/slope)] - 1

AMPLIFICATION



• 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.

SYBR Green Validation

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

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

AMPLIFICATION


Amplicon Design

• 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

AMPLIFICATION


Probe Based Assays

• 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.

AMPLIFICATION


TaqMan Design

• 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

AMPLIFICATION


• Target Sequence

• 2nd Structure Analysis

• Think Small

• Watch out for primer dimers

• Test in Real life conditions

NCBI HomePage

Stacking

No-Web

Next

Primer Design

AMPLIFICATION


• Free resource

• Blast sequence

• Stack sequence

Primer Design

AMPLIFICATION


http://www.ncbi.nlm.nih.

gov/BLAST/Blast.cgi

Primer Design

AMPLIFICATION


• Target Sequence


• Think Small



Mfold – Dr. Zuker

No-Web

Next

Primer Design

AMPLIFICATION


Amplicon Secondary Structures

• Bad location for primers

http://bioinfo.math.rpi.edu/mfold/applications

• Good location for primers

AMPLIFICATION


Reverse Primer B

11110110

Forward

Primer Reverse Primer Reverse Primer

AA

Reverse primer A

ηηηη = 66.3 %

Reverse primer B

ηηηη = 95.8 %

Primer Design

AMPLIFICATION


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

Select target sequence:

Primer Design

AMPLIFICATION


55o C

Dr. Michael Zuker’s mFold

http://www.bioinfo.rpi.edu/

applications/mfold/old/dna/

Primer Design

AMPLIFICATION


60o C




Primer Design

AMPLIFICATION


65o C




Primer Design

AMPLIFICATION


• Target Sequence


• Think Small



Primer Design

AMPLIFICATION


Remember: Keep things as simple and easy as possible

Primer Design

AMPLIFICATION


• Target Sequence


• Think Small



Primer 3

Beacon Designer

No-Web

Next

Primer Design

AMPLIFICATION


• Designs Primers

• Designs Internal Oligos

• Provides multiple outputs

• Free

Web software provided by Steve Rozenand Whitehead Institute for Biomedical Research. http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

Primer Design

AMPLIFICATION


Avoid Primer

Dimers!!

http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_help.cgi#PRIMER_SELF_END

Primer Design

AMPLIFICATION


• Target Sequence


• Think Small



Primer Design

AMPLIFICATION


No resolution below 2000 No resolution below 2000

copiescopies

r = 0.957r = 0.957

ηηηηηηηη = 153%= 153%

ILIL--1b plasmid with SYBR detection1b plasmid with SYBR detection

55--fold dilution series: fold dilution series:

10,000 to 16 copies10,000 to 16 copies

Non validated primers

AMPLIFICATION


No Template400 copies

10,000 copies 2,000 copies


AMPLIFICATION


Same primers with a specific probeSame primers with a specific probe

Poor resolutionPoor resolution

Poor replicatesPoor replicates

ηηηηηηηη = 71%= 71%


AMPLIFICATION


After primer reAfter primer re--design to design to

eliminate primereliminate primer--dimersdimers

r = 0.999r = 0.999

ηηηηηηηη = 91.3%= 91.3%


AMPLIFICATION


Beacon Designer

AMPLIFICATION


RTPrimerDB

Assay Optimization and Validation

AMPLIFICATION


dynamic thermal gradient

Fast Assay optimization

AMPLIFICATION


Gradient Master Mix

• 180 ul 2X iQ SYBR Supermix

• ul forward primer (200nM final)

• ul reverse primer (200nM final)

• ul DNA or cDNA

• ul H20

---------

• 360 ul total

16 wells + 2 (extras) @ 20 ul � 360 ul total

Vortex!


AMPLIFICATION


dynamic thermal gradient

6oC Below

10oC Above


AMPLIFICATION


Melt curve


AMPLIFICATION


Amplification

}Real annealing range


AMPLIFICATION


Proper annealing conditions translates into better uniformity

AMPLIFICATION


Example of 12 replicates

AMPLIFICATION


Validation

AMPLIFICATION


1/3

Blank

1/3 1/3 1/3

1.11 ng/2ul

0.37 ng/2ul

0.12 ng/2ul10.0 ng/2ul

3.33 ng/2ul

Validation

AMPLIFICATION


Validation

AMPLIFICATION


T

Control

Sample A

Sample B

Validation

AMPLIFICATION


Validation

AMPLIFICATION


Test reaction product

ββββ−−−−Actin ODC AZI OAZ

50 bp

100 bp

200 bp

2 kb

Validation

AMPLIFICATION


• In a Hurry?

AMPLIFICATION


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!

AMPLIFICATION


NF1

NF1

NR2

NR2NR1

NF2

NF1

NR1

NF2

NR1

NF2

NR2

In a hurry?

Actin

AMPLIFICATION


NF2

NR2

NF1

NR2NF2

NR1

NF1

NR1

Selecting the best primer set

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

Good Primers

AMPLIFICATION


• Questions?

• Thank You!

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