Optimize Your qpcr and RT-qPCR Assays with Careful Planning and Design

Optimize Your qpcr and RT-qPCR Assays with Careful Planning and Design Nadine Nassif, January 2013

Outline Real-Time PCR overview / Understanding qpcr data qpcr detection strategies: dye-based versus label-based Assay design o Considerations for RT-qPCR Identifying key challenges in the qpcr workflow o Amplification inhibitors o Sample integrity Increasing throughput o Multiplexing o FAST cycling 2

Real-Time PCR (quantitative PCR, qpcr) Detection of Product at Each Cycle Amplification and detection occur together Sensitive Wide dynamic range High throughput capability Multiplex capability Requires minimal sample Requires specialized instrumentation Many manufacturers, many models Hardware differences determine reporter compatibility, multiplexing capability, cost ABI 7500 Real Time System Bio-Rad MyiQ2 3

How does Real-Time PCR compare? More Sensitive with Better Quantitation End-point PCR: Requires minimal sample Only standard thermal cyclers are required Analysis is not high-throughput Quantitation is difficult Microarrays: Fluorescently labeled cdna is hybridized to probe sequences arrayed on a solid surface. Very high throughput; most useful when screening many targets of interest Relative quantitation possible, not as sensitive as qpcr Requires more sample, specialized equipment 4

Real-Time PCR Detection Chemistries Two Common Choices Available A fluorescent reporter is used to detect product formation Two general types: dsdna binding dye Labeled primer or probe or lifetechnologies.com 5

Real-Time PCR Detection Chemistries Easy-to-Use Fluorescent Dye-based Detection DNA-binding dyes bind specifically to the minor groove of dsdna SYBR Green, BRYT Green Free dye has a very low fluorescence Denaturation Denature Annealing Anneal Extension Extend Bound dye has a high fluorescence As more PCR product is produced, the fluorescent signal increases Standard PCR primers are used (no need to order special labeled primers) PCR artifacts can produce non-specific fluorescence

Newer Dyes Offer Improved Detection Sensitivity BRYT Green is a new dsdna binding dye developed by Promega Excitation/emission similar to SYBR Green I (detected using same filters) Greater change in fluorescence = better sensitivity 7

Real-Time PCR Detection Chemistries Highly Specific, Label-based Detection Label-based qpcr uses a labeled primer or probe to detect the amp product. Primer or probe is synthesized with a fluorescent reporter Product formation alters the fluorescence of the reporter Probe allows for specific detection of target sequence; detects only amplification product containing probe sequence Many variations on label-based qpcr chemistry, including: Hydrolysis probe (e.g., TaqMan ) Hybridization probe (e.g., HybProbe) Hairpin probe (e.g., Molecular Beacons) Labeled primer (e.g., Plexor, Scorpions ) 8

Hydrolysis Probe qpcr 2 PCR primers (unlabeled) 1 Probe Denature hv R Q FRET o Labeled with reporter and quencher Primers and probe anneal to template Anneal R Q During extension, 5 nuclease activity of Taq degrades the probe o o Intact probe -> FRET occurs (quenching) Degraded probe -> reporter un-quenched hv R Fluorescence is proportional to the amount of product Extend Q 9

Label-based Methods Enable Multiplexing dsdna-binding dyes only work in monoplex reactions. o Measuring a gene-of-interest transcript and a normalization target requires two separate amplification reactions Label-based chemistry enables amplification of both target and reference gene in the same well, each detected with a differentially labeled probes. o Allows for more data per plate o Gene-of-interest and reference transcript are amplified in the same well, using same template; reduced variability o Multiplexing conserves master mix reagents as well as RNA / cdna sample 10

How to Choose Between Dye-based and Hydrolysis Probe Detection Dye-based Detection Strengths Reduced cost Melt curve analysis is possible Weaknesses Reporter is not sequence-specific Cannot multiplex Hydrolysis Probe Detection Strengths Specific hybridization between probe and target is required to generate signal Multiplexing is possible Weaknesses More expensive Assays are more complicated to design Cannot perform melt curve analysis Pre-designed assays available 11

Interpreting Real-Time PCR Data

Amplification Curve: Shows Accumulation of Product as PCR Progresses baseline initial reporter fluorescence, measured before product formation can be detected exponential phase stage of reaction when product is doubling with each cycle plateau phase stage of reaction when rate of product formation is diminishing Linear scale Plateau Baseline Exponential 13

Amplification Curve: Shows Accumulation of Product as PCR Progresses baseline initial reporter fluorescence, measured before product formation can be detected exponential phase stage of reaction when product is doubling with each cycle plateau phase stage of reaction when rate of product formation is diminishing Log scale Plateau Exponential Baseline 14

Amplification Curve: Shows Accumulation of Product as PCR Progresses threshold the fluorescence level where product can be distinguished from background C q the cycle number at which amplification can be detected by change in fluorescence ΔR n the change in signal during amplification (compared to baseline) C q ΔR n Detection Threshold set above baseline, within exponential region of amp curve 15

Passive Reference A Useful Normalization Tool A passive reference can be used for normalization of amplification curves. Free dye, not associated with amplification Detected in a different color channel than the amplification product Benefit: Normalization to passive reference dye can help control for variation in signal collection across the plate; reduces instrument noise Potential Drawback: Uses a color channel Passive reference is optional 16

qpcr Data May Include a Dissociation (Melt) Curve Melt analysis occurs after amplification is complete Reaction is heated slowly, with signal measured continually Denaturation is detected by a sudden change in fluorescence Melt Peak (T m ) 17

T m is a Function of Amplicon Sequence and Length Dissimilar melt peaks may indicate contamination, non-specific priming, or primer-dimer artifacts Melt analysis helps to verify the specificity of the amplification reactions o One product (single T m ) versus multiple products (multiple melt peaks or shoulders) o Expected T m? o Quality control against false positives Specific amp products Specific T m Artifact T m Late C q in NTC 18

Not All qpcr Chemistries Can Generate Melt Data dsdna binding dye: Signal is generated when dye binds to dsdna Denaturation results in dissociation of dye from product, so signal is reversible Melt analysis is possible Hydrolysis Probe: hv R Q Signal is generated when 5 nuclease activity degrades the probe, separating the quencher from the fluorophore Because the probe is destroyed, the signal generation is NOT reversible Melt analysis is NOT possible 19

Interpreting C q Values Absolute (Standard Curve) Quantitation Requires a standard sample of known concentration A standard curve is generated, and used to determine template quantity in unknown samples C q values of unknown samples are compared to C q values of a standard curve Relative Quantitation Determining relative template levels in test samples versus control samples by comparing C q values Can also normalize the transcript of interest to a reference gene (e.g., GAPDH, beta-actin, 18S rrna) 20

Absolute Quantitation Using a Standard Curve Slope: -3.373 R 2 : 0.9995 Efficiency = 97.9% Standard curve is generated by plotting C q vs (log) concentration for known sample Concentrations of unknown samples are extrapolated from the standard curve, based on C q Amplification efficiency is also derived from the standard curve (function of slope) 21

Amplification Efficiency A Measure of PCR Reaction Quality If amplification is 100% efficient, product doubles with every cycle Slope: -3.373 R 2 : 0.9995 Efficiency = 97.9% Slope of -3.3 = 100% efficiency Ideal range: ± 5% Acceptable range: ± 10% Efficiency = 10 1 ( ) slope -1 100% 22

Relative Quantitation Relative quantitation is based on the premise that the amount of product doubles with each cycle. This relationship also works in reverse: two samples with a C q difference of 1 would have a two-fold difference in starting template amount. o Can be used to determine relative template levels in test samples vs. controls This is only accurate if amplification efficiency is 100%. o Assay validation would include standard curves to calculate efficiency o If amplification efficiency is less than 100%, then calculations can be adjusted to correct for the decreased efficiency Pflaffl, MW (2001). A new Mathematical Model for Relative Quantification in Real-Time RT-PCR. Nucleic Acids Res 29(9):e45. Livak, KJ and Schmittgen, TD (2001). Analysis of Relative Gene Expression Data Using Real Time Quantitative PCR and the 2 -ΔΔCT Method. Methods 25: 402-408. 23

Relative Quantitation Gene of interest: LIN28 Reference gene: GAPDH Differentiation 10 day Differentiation 30 day Stem Cell Differentiated control ΔC q = (C q experimental sample) (C q control sample) ΔΔC q = (ΔC q gene of interest) (ΔC q reference gene) 24

Assay Design Considerations

Detection of RNA Transcripts Use a Combination of Reverse Transcription & qpcr RT-qPCR: Reverse Transcription followed by qpcr amplification. Amplification of a specific RNA molecule into cdna followed by DNA-based PCR amplification. RT-qPCR is a powerful method of gene expression analysis. Determine the presence or absence of a transcript Quantitate mrna expression levels (transcript abundance) 26

2-Step RT-qPCR RT and qpcr Steps Performed in Different Tubes First Step: First strand synthesis Reverse transcriptase is used to generate a cdna copy of an RNA template Either total RNA or mrna can be used as a template Can be performed with random primers, oligo (dt), or a combination of both Second Step: Amplification and quantitation A portion of the cdna is used as the template for real-time amplification Gene-specific primers and thermostable DNA polymerase are added Thermal cycling with the gene-specific primers results in amplification of sequence of interest Product accumulation is monitored at EACH cycle of the amplification 27

Reverse Transcription The RT System You Choose Makes a Difference GoScript Reverse Transcriptase & Kits M-MLV reverse transcriptase Proprietary & optimized buffers Key Features: Efficiently transcribes long mrnas Improved synthesis through RNA secondary structure Performs better in the presence of inhibitors such as ethanol Target mrna 5 AAA n Oligo dt Primed cdna 5 AAA n PCR Primers Random Primed cdna 5 AAA n Better cdna Length Ammerschläger, M., et al. Lining Up the Scripts: Reverse Transcriptase Comparison Study O = Oligo dt primers R = Random primers 28

2-Step RT-qPCR cdna Primer Choice Excluding random primers from cdna synthesis reactions where total RNA is used can eliminate production of ribosomal cdna Priming with oligo (dt) alone may introduce some 3 bias However, cdnas produced by priming with oligo (dt) alone can be over 1,000 nucleotides long; should be sufficient for good representation of the transcripts Primer studies using microarrays: cdna was generated with oligo (dt) alone and with oligo (dt) + random primers Hybridization patterns were analyzed; approximately 4% of hybridization events showed significant deviation Whichever primer mix is used: use it consistently! 29

1-Step RT-qPCR RT and qpcr Steps Performed in the Same Tube 1-Step RT-qPCR combines first-strand synthesis with real-time amplification, in a single reaction tube. Gene-specific primers Reverse transcriptase Thermostable DNA polymerase RNA template First-strand synthesis: gene-specific primers + reverse transcriptase generate the cdna. Thermal cycling: gene-specific primers + DNA polymerase result in amplification of the sequence of interest. 30

Choosing an RT-qPCR Strategy Simplicity vs. Multiple Target Analysis 1-Step RT-qPCR: Reverse transcription and qpcr performed in a single tube. Gene-specific primers used for both reverse transcription and amplification 1 aliquot of RNA sample is consumed for 1 qpcr reaction May be the most sensitive approach 2-Step RT-qPCR: Reverse transcription followed by qpcr in separate tube. Oligo (dt) and/or random primers are used for reverse transcription Gene-specific primers are used for qpcr 1 aliquot of RNA can create a pool of cdna that can be archived for future use and utilized for multiple qpcr analyses o Analysis of different target sequences o Technical replicates 31

Primer Design Considerations Smaller amplicons (< 150bp) are more likely to give better efficiency For RNA specificity: amplicon (or primer) should span exon:exon junction Evaluating primer design: Check for specificity in silico with BLAST search o Confirm target sequence is from the correct organism / reference sequence o Alternative splicing? Pseudogenes? Species- or strain-specific variations? o If there are matches to unintended sequences, evaluate percent identity and predicted T m of interaction Experimental validation o Test primers using a positive control sample dilution series o Evaluate amplification efficiency and dynamic range 32

Primer Design Resources Pre-designed qpcr Primers: Primer Bank: http://pga.mgh.harvard.edu/primerbank/ RTPrimerDB: http://www.rtprimerdb.org/ Primer design software: Primer3: http://frodo.wi.mit.edu/primer3/ Primer-BLAST: http://www.ncbi.nlm.nih.gov/tools/primer-blast/ Primer Design Algorithms, Databases & Resources: Gene Quantification: http://www.gene-quantification.info/ Sequence Resources: Gene Name + Aliases Reference Sequence 33

Considerations for Optimizing qpcr Assays Check Key Parameters for Best Results Magnesium concentration o Higher Mg 2+ concentrations tend to favor hybridization o Excessive MgCl 2 can promote non-specific product formation Thermostable DNA polymerase o Hot-start polymerases reduce non-specific amplification o Enzyme concentration affects performance Primer / Probe concentration o Titrations should be performed to ensure optimal results o For gene expression assays, primer / probe concentration may need to be adjusted based on target abundance Cycling Parameters o Annealing temperatures o Hold times 34

Key Control Reactions Ensure Quality Results No Template Control (NTC) o There should be no amplification product detected in NTC reactions Detected product indicates contamination with amplicon or starting template o Detection of product in NTC reactions defines a limit of detection Positive Control o Known concentration, free of inhibitors o May have an expected C q and T m No Reverse Transcriptase Control o If RT is absent during cdna synthesis, then no real-time amplification should be observed o Demonstrates how much amplification is due to DNA contamination 35

Challenging Variables in qpcr Assays: Template Quantity, Purity, and Integrity

Template Quantity Input Template Amount Affects Final Results Amount of RNA required to detect a transcript of interest will depend upon the abundance of that RNA transcript in each sample. o A high-copy RNA transcript may be reliably detected in as little as 10pg o A low-copy RNA transcript may require more than 100ng Too much template can inhibit qpcr. o Excess nucleic acids can bind Mg 2+ o When cdna is added to amplifications without intermediate purification, RT reaction components (salts, etc.) are coming along for the ride o Increasing the amount of template in each reaction can also increase the amount of sample contaminants that are introduced 37

Template Purity Contaminants Can Impact qpcr Assays Endogenous cellular contaminants o o o Proteins (including nucleases) Carbohydrates gdna contamination in RNA templates Contaminants introduced by the environment o Hematin o Tannic acid (leather), indigo dye (denim) o Humic acid o Bile o CaCl 2 o Urea Contaminants introduced during nucleic acid purification o Chaotropes (e.g., guanidine) o Detergents (e.g., SDS) o Alcohols o Organic solvents o Chelators (e.g., EDTA) 38

Template Purity Absorbance Reading May Detect Contaminants flychip.org.uk 39

Template Purity Inhibitors May Be Present in Starting Samples Environmental samples can be challenging with qpcr analysis o Food samples, fecal samples, soil, wood, plant material, textiles Inhibitors can be introduced during nucleic acid purification o Chaotropes, alcohols, chelators, detergents, solvents Some qpcr master mixes will perform better in the presence of inhibitors 40

Template Purity GoTaq System Exhibits Resistance to Hematin GoTaq Probe qpcr (Promega) Vendor #1 Hematin 0 µm 20 µm 30 µm 50 µm Hematin 0 µm 20 µm 30 µm 50 µm 41

Template Purity GoTaq System Exhibits Resistance to Humic Acid GoTaq Probe qpcr (Promega) Vendor #2 Humic Acid 0 ng 200 ng 300 ng 400 ng Humic Acid 0 ng 200 ng 300 ng 600 ng 400 ng 600 ng 42

Template Purity GoTaq System Exhibits Resistance to Phenol GoTaq Probe qpcr (Promega) Vendor #3 Phenol 0% 0.5% 1.0% 1.5% Phenol 0% 0.5% 2.0% 1.0% 1.5% 2.0% 43

Template Integrity Fragmented Samples May Affect qpcr Results Some sample types are more likely to be degraded Samples exposed to nucleases, heat and UV light FFPE samples: cross-linking introduced by the fixation and embedding process results in nucleic acids that are characteristically partially degraded When designing downstream amplification assays, best results will be achieved when targeting regions of 200 nucleotides or less Options exist for assessing template integrity, including: Gel analysis Agilent Bioanalyzer (Agilent Technologies) Fragment Analyzer (Advanced Analytical) 44

Assessing Sample Integrity by Gel Analysis Old School, but It Works gdna Incubated +/- DNase I - 23,130bp 9416bp 6557bp 2322bp 1500bp 1000bp 564bp 500bp 45

Assessing Sample Integrity with an Agilent Bioanalyzer High quality total RNA 28S rrna and 18S rrna appear as distinct, intact bands Ratio of 28S:18S bands is approximately 2:1 Poor quality total RNA Degradation of 28S rrna and 18S rrna is apparent Poor 28S:18S ratio 46

Quality Examples for Samples Assessed on an Agilent Bioanalyzer RNA Integrity Number (RIN) RNA electropherogram is analyzed by the software Provides an estimate of total RNA integrity RIN is on a scale: 1 (most degraded) 10 (most intact) agilent.com 47

Assessing Sample Integrity with the Fragment Analyzer gdna Quality: More intact More degraded 48

Multiplexing qpcr Assays

Label-based qpcr Enables Multiplexing Label-based qpcr enables multiplexing via differential labeling of PCR primers or detection probes. Advantages: o Allows for more data per plate; higher throughput o Gene-of-interest and reference transcript are amplified in the same well, using same template; reduced variability o Multiplexing conserves master mix reagents as well as precious RNA or cdna sample Possible Concerns: o Multiplexing can affect amplification efficiency 50

Key Considerations When Multiplexing How many amplicons can be multiplexed into a single reaction? o Defined by instrument capability o Excitation source (laser, LED, halogen lamp) + emission filters o Some older instruments were limited to two dye channels o Most now capable of 4 or 5 dyes (some up to 6) Design multiplex combinations so the weakest amplicon is detected in the strongest dye channel Design primers with similar annealing temperatures Assess amplification efficiency in monoplex reactions, and then assess again in multiplex 51

Multiplex Assay Design Assess Monoplex Amplification Efficiency Target: TNNT2 Dye: FAM Target: GAPDH Dye: JOE Slope: -3.341 R 2 : 0.9993 Efficiency: 99.2% Slope: -3.453 R 2 : 0.9991 Efficiency: 94.8% 52

Multiplex Assay Design Assess Duplex Amplification Efficiency Target: GAPDH Dye: JOE Slope: -3.408 R 2 : 0.9994 Efficiency: 96.5% Target: TNNT2 Dye: FAM Slope: -3.319 R 2 : 0.9990 Efficiency: 100.1% 53

Optimize Multiplexed qpcr by Adjusting the Same Key Parameters in Monoplex qpcr The same considerations apply for multiplexed qpcr as for monoplexes. Magnesium concentration Enzyme concentration Primer / Probe concentration Cycling Parameters 54

Standard Versus FAST Cycling

Standard Versus FAST Cycling Fast Cycling Increase Sample Throughput Standard versus fast cycling is determined by: Hot Start Taq activation time can vary from 20 seconds to 15 minutes Thermal cycling times Instrument ramp rates Step # Cycles Temperature Standard Cycling Time FAST Cycling Time GoTaq Activation 1 95 C 2 minutes 20 seconds - 2 minutes Denaturation 95 C 15 seconds 3 seconds 40 Annealing/Extension 60 C 1 minute 30 seconds Advantage: Faster cycling means higher throughput Possible Concern: May result in lower sensitivity or increased variability. 56

GoTaq Probe qpcr Master Mix: Robust amplification with standard and fast cycling GoTaq Probe: standard cycling GoTaq Probe: FAST cycling AB 7500 AB 7500 FAST Slope: -3.385 R 2 : 0.9986 Efficiency: 97.4% Slope: -3.386 R 2 : 0.9988 Efficiency: 97.4% Cycling method 100ng 10ng 1ng 0.1ng 10pg 1pg 0.1pg GoTaq standard 17.3 20.7 23.9 27.3 30.7 34.3 36.0 GoTaq FAST 17.6 21.2 24.4 27.9 31.2 34.6 36.5 57

GoTaq Probe qpcr Master Mix: Robust amplification with standard and fast cycling Vendor #2 fast master mix: FAST cycling GoTaq Probe: FAST cycling AB 7500 FAST AB 7500 FAST Slope: -3.359 R 2 : 0.9983 Efficiency: 98.5% Slope: -3.386 R 2 : 0.9988 Efficiency: 97.4% Cycling method 100ng 10ng 1ng 0.1ng 10pg 1pg 0.1pg Vendor #2 FAST 17.7 21.0 24.5 27.8 31.2 34.4 37.7 GoTaq FAST 17.6 21.2 24.4 27.9 31.2 34.6 36.5 58

qpcr Resources

MIQE is a Valuable Reference Minimum Information for Publication of Quantitative Real-Time PCR Experiments MIQE provides specifications for the minimum information that must be reported for a qpcr experiment to ensure relevance, accuracy, correct interpretation and reproducibility. MIQE paper and checklist: http://www.rdml.org/miqe.php 60

General qpcr Resources General Gene Quantification: www.gene-quantification.info/ Promega.com: FAQ for RT-qPCR A Z of quantitative PCR (Editor: S.A. Bustin ), International University Line, La Jolla, CA. Webinars Maximize Your Reverse Transcription-qPCR (RT-qPCR) Assays -- Carl Strayer, PhD Optimizing RNA Expression Analysis from Start to Finish -- Doug Wieczorek, PhD Technical support http://www.promega.com/support/customer-and-technical-support/ 61

Promega Products for qpcr and RT-qPCR Sample Collection & Processing Nucleic Acid Extraction Nucleic Acid Quantification & Assessment Reverse Transcription qpcr Data Analysis qpcr and RT-qPCR GoTaq qpcr and RT-qPCR Master Mixes dye-based detection with BRYT Green GoTaq Probe qpcr and RT-qPCR Master Mixes for label-based detection Reverse Transcription GoScript Reverse Transcription System Nucleic Acid Extraction / Purification ReliaPrep gdna or Total RNA Purification Kits Blood, tissue, cells, FFPE samples Maxwell 16 Purification Kits for use with the Maxwell 16 instrument 62

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