Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar Series Part 1
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This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
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Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar Series Part 1
- 1. Sample to Insight Introduction to Real-Time Quantitative PCR (qPCR) Webinar-relatedquestions: QIAwebinars@qiagen.com Technical Support: BRCsupport@qiagen.com
- 2. Sample to Insight Welcome to our four-part webinar series on qPCR 2 qPCR technology overview, applications, data analysis and service solutions ⢠Part 1: Introduction to Real-Time PCR (Q-PCR / qPCR/ qrt-PCR) ⢠Part 2: Advanced Real-Time PCR Array Technology â Coding and Noncoding RNA Expression Analysis ⢠Part 3: PCR Array Data Analysis Tutorial ⢠Part 4: Accelerate your Discovery with QIAGEN Service Solutions for Biomarker Research
- 3. Sample to Insight Legal disclaimer QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Servicesor your local distributor.
- 4. Sample to Insight Aspects of a good real-time PCR assay4 Agenda 4 Real-time PCR overview and applications1 Steps involved in real-time PCR2 Real-time PCR reporter chemistries3 Data and analysis5
- 5. Sample to Insight How does qPCR work? Question: How far apart are the two cars? The cars race at same speed to finish line ⢠As Car 1 crosses finish line, calculate time for Car 2 to finish ⢠Calculate the difference in starting position mathematically (distance = rate à time) Finish line Car 2 Car 1
- 6. Sample to Insight How does qPCR work? Finish line Car 2 Car 1 Question: How far apart are the two cars? ⢠Many cars â how to differentiate cars of interest
- 7. Sample to Insight How does qPCR work? Finish Line Car 2 Car 1 Question: How far apart are the two cars? ⢠The cars race at same speed to finish line ⢠As Car 1 crosses finish line, calculate time for Car 2 to finish ⢠Calculate difference in starting position mathematically (distance = rate à time)
- 8. Sample to Insight Seminar topics 1. What is qPCR? Applications and workflow 2. qPCR for gene expression: What is the change in gene expressionduring differentiation? 3. Factors criticalfor a successful qPCR assay 4. RNA purity and integrity 5. Reverse transcription 6. qPCR in action 7. Reporter chemistries 8. Characteristics of a good qPCR assay 9. Analyzing qPCR curves 10. Data and analysis
- 9. Sample to Insight What is qPCR? Applications and workflow What does real-time qPCR stand for? ⢠Quantitative polymerase chain reaction(qPCR) is a sensitive and reliable method for detection and quantification of nucleic acid (DNAand RNA) levels. ⢠It is based on detection and quantification of fluorescence emitted from a reporter molecule in real time. ⢠Detectionoccurs during the accumulation of the PCR product with each cycle of amplification. This allows for monitoring of the PCR reactionduring the early and exponential phases where the first significant increase in the amount of PCR product correlates to the initial amount of target template.
- 10. Sample to Insight What is qPCR? Applications and workflow RNA DNA Applications for qPCR ⢠Gene expression profiling analysis ⢠miRNA expression profiling analysis ⢠SNP genotyping and allelic discrimination ⢠Somatic mutation analysis ⢠Copy number detection / variation analysis ⢠Chromatin IP quantification ⢠DNA methylation detection ⢠Pathogen detection ⢠Viral quantification
- 11. Sample to Insight What is qPCR? Applications and workflow RNA (total, mRNA, small RNA) Sample QC cDNA Sample DNA Sample QC Set up real-time PCR Set up instrument and thermocycling Data output and analysis SYBR /probe Assay design Assay optimization Workflow: a brief look
- 12. Sample to Insight What is qPCR? Applications and workflow RNA DNA Applications for qPCR ⢠Gene expression profiling analysis ⢠miRNA expression profiling analysis ⢠SNP genotyping and allelic discrimination ⢠Somatic mutation analysis ⢠Copy number detection / variation analysis ⢠Chromatin IP quantification ⢠DNA methylation detection ⢠Pathogen detection ⢠Viral quantification
- 13. Sample to Insight qPCR for gene expression: application example hMSC Osteogenesis â Day 16 Neurogenesis â 72 hr T1 T2 T3 T4 T1 T2 T3 T4 Gene expression changes during differentiation ⢠Differentiationprotocol ⢠Collect total RNAat different time points ⢠Measure 1 HKG and 1 GOI (TNFa) ⢠Repeat experiment three times (biological replicates)
- 14. Sample to Insight qPCR for gene expression Total RNA Sample QC cDNA Set up real-time PCR Thermocycling Data analysis SYBR Assay design Assay optimization Workflow: gene expressionprofiling
- 15. Sample to Insight Factors critical for a successful qPCR assay ⢠DNA or RNA sample preparation â template quality o Appropriate sample prep kits / reagents o Inhibitors can compromise RT or PCR ⢠Reverse transcription to convert RNA to cDNA o Choose RT kits â Type of RT â Which type of primers â Controls? ⢠Assay design: chemistry, specificity, PCR efficiency, throughput and cost o Choose validated assay or need to validate our own? ⢠Running PCR o Commercial master-mix or make own (primer, probe, master-mix) ⢠Data analysis tool o User-friendly o Streamlined data analysis module
- 16. Sample to Insight RNA purity and integrity RNA Isolation: ⢠QIAzol? ⢠Column-based method (RNeasy?) ⢠Both: efficient lysis and inhibition of RNases; molecular grade RNA ⢠miRNA? Use a kit specific for miRNA and mRNA miRNeasy Mini Kit QIAzol: Phenol / guanidine-based lysis Column cleanup: Molecular biology grade RNA Archive miRNA for next project Instant inactivation of RNases Instant end of biological activities
- 17. Sample to Insight RNA purity and integrity Purity/quantity: Spectroscopic: measure 260/280 and 260/230 ⢠OD260 is used to calculate amount of nucleic acid ⢠260/280 ratio (typical minimum value 1.8-2.0) ⢠260/230 ratio (typical minimum value 1.7) o Low ratio may indicate a contaminant; protein, QIAzol, carbohydrates, glycogen o Absorbance measurements do not show integrity of RNA Integrity: Denaturing RNA agarose gel ⢠Usually through ribosomal bands QIAxcel / bioanalyzer: ⢠Capillary electrophoresis ⢠Automate RNA integrity analysis ⢠RNA integrity analysis number
- 18. Sample to Insight Factors critical for a successful qPCR assay ⢠One tube reaction One-step PCR ⢠Two separate reactions ⢠RT reaction ⢠qPCR reaction Two-step PCR A. Templates: ⢠RNA o Starting amount ~10-1000 copies of RNAper qPCR assay o For a low-expressed gene, need 10 ng equivalent of RNA per reaction o Want to start with about 100 pg to 1 Οg RNA ⢠Reverse transcription o One-step or two-step reaction B. Primers / probes C. Master-mix o DNA polymerase o Mg++ o dNTPs o Buffer o Passive reference dye qPCR components
- 19. Sample to Insight Reverse transcription Reverse transcription: used to make a cDNA copy of RNA Reagents: ⢠Reverse transcriptase â many different kinds ⢠dNTPs ⢠Buffers for RT ⢠Primers o Random pentamers or hexamers o Oligo-dT o Both ⢠Control RNA to monitor reverse transcription kit? ⢠Important notes: o Ensure RT reaction is linear o Do not try to reverse transcribe too much RNA o Sensitivity of qPCR step is dependent on good RT reaction o Monitor RT reaction to ensure equal RT efficiency across all samples
- 20. Sample to Insight Aspects of a good real-time PCR assay4 Agenda 20 Real-time PCR overview and applications1 Steps involved in real-time PCR2 Real-time PCR reporter chemistries3 Data and analysis5
- 21. Sample to Insight qPCR in action DNA template (ss or ds) What is in a PCR Reaction? PCR = Polymerase Chain Reaction Exponential amplification of DNA in single tube All reagents in excess (non-limiting) Components: ⢠Thermostable polymerase ⢠dNTPs ⢠Primers ⢠Template Polymerase dNTPs Primers (2)
- 22. Sample to Insight qPCR in action DNA template (ss or ds) 1. Heat denature template (~95°C) 2. Annealing (~60°C) 3. Extension (~60°C) 4. Repeat (~95°C) Polymerase dNTPs Primers (2)
- 23. Sample to Insight qPCR in action DNATemplate (ss or ds)Heat denature 1. Heat denature template (~95°C) 2. Annealing (~60°C) 3. Extension(~60°C) 4. Repeat (~95°C) Polymerase dNTPs Primers (2)
- 24. Sample to Insight qPCR in action DNA template 1. Heat denature template (~95°C) 2. Annealing (~60°C) 3. Extension (~60°C) 4. Repeat (~95°C) Polymerase dNTPs Primers (2)
- 25. Sample to Insight qPCR in action DNA template (ss or ds) Polymerase Polymerase dNTPs Primers (2) Polymerase 1. Heat denature template (~95°C) 2. Annealing (~60°C) 3. Extension (~60°C) 4. Repeat (~95°C)
- 26. Sample to Insight qPCR in action DNA template (ss or ds) 1. Heat denature template (~95°C) 2. Annealing (~60°C) 3. Extension (~60°C) 4. Repeat (~95°C) Polymerase Polymerase Polymerase dNTPs Primers (2)
- 27. Sample to Insight qPCR in action DNA template (ss or ds) Polymerase Polymerase Polymerase dNTPs Primers (2) 1. Heat denature template (~95°C) 2. Annealing (~60°C) 3. Extension (~60°C) 4. Repeat (~95°C)
- 28. Sample to Insight qPCR in action DNA template (ss or ds) Polymerase dNTPs Primers (2) 1. Heat denature template (~95°C) 2. Annealing (~60°C) 3. Extension (~60°C) 4. Repeat (~95°C)
- 29. Sample to Insight qPCR in action How do you make this a quantitative PCR? Measure DNA amount at end of each cycle to get ratio of DNA or absolute amount (if using a standard) 1. Heat denature template (~95°C) 2. Annealing (~60°C) 3. Extension (~60°C) 4. Measure amount of PCR product 5. Repeat (~95°C) Polymerase dNTPs Primers (2) DNA template (ss or ds)
- 30. Sample to Insight Aspects of a good real-time PCR assay4 Agenda 30 Real-time PCR overview and applications1 Steps involved in real-time PCR2 Real-time PCR reporter chemistries3 Data and analysis5
- 31. Sample to Insight Reporter chemistries Real-time qPCR fluorescence chemistry â DNA binding agents o SYBRÂŽ I dye ⢠Hydrolysis probes o Dual-labeled hydrolysis (TaqmanÂŽ) probe ⢠Others such as hybridization probes o Molecular beacon and ScorpionsÂŽ probes
- 32. Sample to Insight Reporter chemistries: SYBRŽ Green I assay Non fluorescent SYBR I Fluorescent SYBR I SYBR I binds to double-strand DNAbut not single-strand DNA. Little fluorescenceemitted from SYBR I in solution SYBR I upon binding to double-strand DNA emits fluorescence very brightly The SYBR I signal intensities correlate with DNA amplified (amplicon amount) and thus the initial sample input amounts ⢠Simple and costsaving ⢠Highspecificity isrequiredwhen using SYBR Green since SYBR I binds all double-strand DNA (non-specificor primer dimer)
- 33. Sample to Insight Reporter chemistries: understanding kinetics in PCR Plateau 107 106 105 Fluorescencesignal Amplification plot (linear scale) ⢠End-point PCR data collection at plateau (gel analysis) ⢠Reactions start varying due to reagent depletion and decreased PCR efficiencies (enzyme activity, more product competing for primer annealing) ⢠Real-time PCR does early phase detection at the exponential state ⢠Precisely proportional to input amounts
- 34. Sample to Insight Reporter chemistries Hydrolysis-based probe â TaqmanÂŽ probe assay The fluorescence of the reporter dye is suppressed by the quencher Primer binding followed by extension Probe cleavage by Taq to free the reporter dye thus the fluorescence intensity correlates with the initial sample input amounts Taq has 5â 3â exonuclease activity Each amplicon needs a sequence-specific probe (cost and time)
- 35. Sample to Insight Reporter chemistries: understanding kinetics in PCR Plateau 107 106 105 Fluorescencesignal Amplification plot (linear scale) ⢠End-point PCR data collection at plateau (gel analysis) ⢠Reactions start varying due to reagent depletion and decreased PCR efficiencies (enzyme activity, more product competing for primer annealing) ⢠Real-time PCR does early phase detection at the exponential state ⢠Precisely proportional to input amounts
- 36. Sample to Insight Aspects of a good real-time PCR assay4 Agenda 36 Real-time PCR overview and applications1 Steps involved in real-time PCR2 Real-time PCR reporter chemistries3 Data and analysis5
- 37. Sample to Insight Characteristics of a good qPCR assay What factors do you need to address to create a good PCR assay? ⢠Amplification efficiency: exponential phase is 100% (template product doubles with each cycle) ⢠Sensitivity: able to detect down to reasonable quantities of template in one reaction (10-50 copies) â Specificity: one assay, one target (no off-target amplification or primer dimers) ⢠Melting curve analysis â one peak, one product ⢠Agarose gel ⢠Dynamic range: ability to detect genes with varied expression levels â another judge of sensitivity o Ideally 10 to 109 copies ⢠Reproducibility: confidence in your results â enables profiling of multiple genes in the same sample o All lab members get the same results o Technical reproducibility ensures changes seen in results are due to the biology and not sample handling or the technology itself
- 38. Sample to Insight Characteristics of a good qPCR assay: amplification efficiency Amplification efficiency: reliable and accurate experiment Two methods: ⢠Standard curve analysis o X axis â dilution o Y axis â Ct value o Amp. efficiency = (10(â1/slope) â 1) Ă 100 ⢠Single curve analysis o PCR Miner: http://miner.ewindup.info/versi on2 o âDARTâ: www.gene- quantification.de/DART_P CR_version_1.0.xls
- 39. Sample to Insight Characteristics of a good qPCR assay: sensitivity Sensitivity: how many copies can my assay detect? ⢠Important for low-expressed genes or where there is limited sample Two Methods: ⢠Method 1: use primers to make PCR product, T/A clone, grow up, isolate, quantify and use for qPCR reactions ⢠Method 2: use gDNA as template and use mass of gDNA to calculate copy number and assume one target per genome (or actually calculate targets using bioinformatics)
- 40. Sample to Insight Characteristics of a good qPCR assay: specificity Specificity: one target amplified Two Methods: ⢠Melting curve analysis o One peak, one product ⢠Agarose gel o Band at correct size
- 41. Sample to Insight Characteristics of a good qPCR assay: specificity Plot â normalized reporter Melting curve analysis Gene ATm: 77.36°C Gene B Tm: 78.94°C Normalizedfluorescencesignal Temperature 50% fluorescence drop Rn General program steps ⢠Heat to 94°C to denature DNA ⢠Cooling to 60°C to let DNA double strands anneal ⢠Slowly heat (increase temperature 0.2°C/s) while plotting the fluorescent signal versus temperature ⢠As the temperature increases, DNA melts and fluorescent signal should decrease ⢠Significant drop in signal when 50% of DNA melts
- 42. Sample to Insight Characteristics of a good qPCR assay: specificity Plot â 1st negative derivative reporter Temperature âÎF/ÎT(rateofchange) Gene A Tm: 77.36°C Gene B Tm: 78.94°C Single melting curve of each amplicon is required for specificity validation Melting curve analysis
- 43. Sample to Insight Aspects of a good real-time PCR assay4 Agenda 43 Real-time PCR overview and applications1 Steps involved in real-time PCR2 Real-time PCR reporter chemistries3 Data and analysis5
- 44. Sample to Insight Biological replicates are better than technical replicates Biological replicates: three different experiments ⢠Shows variability due to experiment Technical replicates: three different measurements for same step ⢠Shows variability due to pipetting, machine, enzymes, etc.
- 45. Sample to Insight Analyzing qPCR curves: how to define baseline Linear amplification plot Baseline Ct Automated baseline option ⢠Instrument establishes baseline Manual baseline option ⢠Use linear view of the plot ⢠Establish baseline beginning at cycle two and subtracting two cycles from earliest amplification seen ⢠Usually the baseline falls between cycles 3- 15
- 46. Sample to Insight Analyzing qPCR curves: how to define threshold Log view amplification plot ⢠Use log view of amplification plot ⢠Threshold should be higher than baseline (higher than the noise level) ⢠Threshold should be at lower third or half of the linear phase of amplification ⢠Linear phase = exponential phase ⢠Different runs across samples for the same experiments should have the same threshold for comparison
- 47. Sample to Insight Reference gene ⢠Expression level remains consistent under experimental conditions / different tissues ⢠Aimed to normalize possible variations during: o Sample prep and handling (e.g., use the same number of cells from a start) o RNA isolation (RNA quality and quantity) o Reverse transcription efficiency across samples / experiments o PCR reaction setup o PCR reaction amplification efficiencies GOI A in control cells GOI A in drug treatedcells Reference gene B in control cells Reference gene B in drug treatedcells Any changes? Data analysis: housekeeping / reference genes
- 48. Sample to Insight Data analysis: commonly used housekeeping genes
- 49. Sample to Insight Data and analysis 1. Average Ct values for all gene replicates 2. Calculate ÎCt value between GOI and HKG for each experiment 3. Average ÎCt values between experiments (replicates) 4. Calculate ÎÎCt values (ÎCtexperiment â ÎCtcontrol) 5. Calculate fold change (2(âÎÎCt))
- 50. Sample to Insight Data and analysis Normalized Gene Expression Level Target Gene A in control cells Target Gene A in drug treatedcells Reference Gene B in control cells Reference Gene B in drug treatedcells Any changes? âCt = Ct (Target Atreated) â Ct (Ref Btreated) âCt = Ct (Target Acontrol) â Ct (Ref Bcontrol) ââCt = âCttreated â âCtcontrol Normalized target gene expression level = 2(âââCt)
- 51. Sample to Insight Data and analysis: ÎÎCt method â amplification plots Reference GOI GAPDH TNFa ââCt= âCt (TNFÎątreated â GAPDHtreated) â âCt (TNFÎącontrol â GAPDHcontrol) Fold change = 2(âââCt) Ct Ct Ct Ct
- 52. Sample to Insight Data and analysis 1. Average Ct values for all gene replicates 2. Calculate ÎCt value: GOI-HKG 3. AverageÎCt values between experiments (replicates) 4. Calculate ÎÎCt values (ÎCtexperiment â ÎCtcontrol) 5. Calculate fold change (2(âÎÎCt)) TNFa is upregulated 32-fold in treated cells versus control 17.1, 17.2, 17.2 qPCR replicates
- 53. Sample to Insight http://www.qiagen.com/Products/Genes and Pathways/Data AnalysisCenter Overview Page/Data analysis
- 54. Sample to Insight http://www.qiagen.com/Products/Genes and Pathways/Data AnalysisCenter Overview Page/Data analysis tools
- 55. Sample to Insight Topics covered today 1. What is qPCR? Applications and workflow 2. qPCR for gene expression: what is the change in gene expressionduring differentiation? 3. Factors criticalfor a successful qPCR assay 4. RNA purity and integrity 5. Reverse transcription 6. qPCR in action 7. Reporter chemistries 8. Characteristics of a good qPCR assay 9. Analyzing qPCR curves 10. Data and analysis
- 56. Sample to Insight Upcoming webinars: still searching gene by gene? Learn about RT2 Profiler PCRArrays 384-wellCatalogued RT2 Profiler by pathway and disease 4x96 370 Pre-validated qPCR assays with controls
- 57. Sample to Insight Thank you for attending Questions? Ask now or contact Technical Support ⢠Call: 1-800-426-8157 ⢠Email: BRCsupport@qiagen.com For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.
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- Copy: We will start with a brief explanation of what you actually use qPCR for any why. There are generally 2 types; relative real-time PCR, which we will discuss today, and absolute PCR. Relative real-time PCR: You have 2 samples and you want to know what is the difference in these genes in our samples? Ex. Is MIC up/down regulated in a cancer sample Absolute PCR: We use a known standard and we want to know the quantity of something/ how many copies of something is present (ex. Virus in particular volume) Experiment: how far apart are these 2 cars from their initial starting position. Is car 2 three or four times as far away from car 1? What is the difference? She knows she can develop a race with the equation d=RxT. She knows these cars are identical, which is an important aspect of qPCR. We are looking at the same gene in different samples. And if we know that the cars are moving at the same rate, then the difference in the initial distance is proportional to the difference in time. If these cars (or samples) are going at the same rate, then the distance should be proportional to how long it takes car 2 to cross the finish line. In relative real time PCR, we are doing the same thing. We are using a kinetic race of the real time PCR reaction to go through and calculate the difference in our starting analytes. What is real time PCR? Explain what are you trying to achieve? have 2 samples (control, experimental), What is difference? Is gene expression going up or down? 2 samples, same gene, what is relative difference? (car example: scientist cannot see the cars at the different points visually, so she devises an experiment w/ rate*time to calculate initial difference eg. Car race w/ finish line to NY; first car crosses finish line, start a timer to see when second car finishes relative to the first one) qPCR machines do the same, kinetic race. Calculates difference between analytes.
- Copy: Our scientist needs to identify only these 2 cars of interest from all of the other cars out there. If you think about qPCR, you are doing a kinetic race between our 2 samples. Our finish line is the threshold line in our instrument, and instead of a car engine we have the kinetics of our qPCR reaction, and instead of the distance and time, we are talking about the difference in Ct values. (idealized reaction) Actually many cars, needs to differentiate the cars of interest between all of the other cars (specificity). Also, what if one car is lost and unable to see the car (sensitivity; affects data analysis). If one car goes off path (to Canada), or car falls apart and rate changes (efficiency; affects data analysis)? These reactions needs to be efficient (as close to 100% amplification efficiency as possible), specific (each assay to recognize only 1 target), and sensitive (need to recognize an optimal amount of copies, usually around 20 copies).
- qPCR is the engine to race to finish line (threshold). Amplify gene to threshold. As 1 samples crosses threshold, watch other anaylte. Mathematically solve. Look at differences in cycles it takes for 2nd samples to cross finish line (not really time). Look at different starting amounts. qPCR is a kinetic race (specificity, sensitivity, amplification efficiency). Look at difference in Ct value.
- Gene expression plus more! (methylation, quantify viruses or bacteria) Example (pluripotent stem cells experiment) Influencing factors, Factors influencing the performance of a qPCR assay RNA quality (purity/ integrity) RT reaction- critical to make cDNA we need, if compromised it will determine cDNA and affect our sensitivity, if not good cDNA you cannot detect gene of interest qPCR diagram, what is actually happening in your CR tube and how do you go from a regular end-point PCR reaction to a Real-time quantitative PCR reaction. Well talk about how to measure the fluorescence to make this reaction real-time and the main products to use to measure thiss, SYBR green, hydrolysis probes, when to use which Characteristics of GOOD real time PCR assay (validation) Analyzing qPCR curves Some analysis
- (read) gold standard to look at CNV, RNA (mRNA, miRNA, lncRNA (just need a good cDNA copy). It is usually used to validate other methods for gene expression. It is reliable: once you have an optimized assay, its very reliable reaction It is sensitive: we can see down to a single copy if we use digital PCR, typically, if we have 20 copies in a reaction we can see that with a good primer design. (read) have 1 component for thermocycling block component, 1 for exciting molecule and detecting fluorescence; cannot measure individual reaction, but can measure amount of fluorescence, proportional to amount of product made each cycle (end-point is just the amount at the end of a set number of cycles) qPCR has a dynamic linear range, quantitate copies ( 20 to 20*10^7) without dilutions (ELISA, western blots) and gels (read) measure the reaction at the right time. Set threshold where we have non-limiting reaction (exponential phase) Look at curves at linear and log scale. Allows good data from reaction. No rate-limiting steps, then in theory the reaction is moving at 100% . Low threshold could be background noise, high threshold
- Can be very powerful research/ diagnostic instrument RNA â routine; start with RNA, convert this to cDNA and detect whatever expression we want to look at DNA â SNP Genotyping & allelic discrimination (read) Somatic Mutation Analysis- individual mutation, insertions, deletions Copy Number Detection/Variation Analysis- copy number of individual genes or loci Chromatin IP Quantification- Chromatin Immunoprecipitation experiment, which is a powerful method to identify where protein and DNA are binding together on regions on gDNA, also for understanding histone code, insight into insight to confirmation of chromatin structure (epigenetic mechinisms) DNA Methylation Detection- gNDA to Bisulfite conversion to methyl-sensitive PCR or restriction enzymes to cleaev DNA based on methylation paterns and identify that with PCR Pathogen Detection â can use relative, or use absolute and incorporate standard, which we know how many copies we have so that we know the stoiceometry of what were trying to measure. Viral Quantification -
- So how would you implement a qPCR experiment into your lab, what would your basic process flow look like and what variables do we want to control to get accurate and reproducible data? There are a lot of ways to a result based on your starting sample and biological question is. Of course, you start with your sample and if you are using, RNA âyou can start with total, mRNA, or small RNA (miRNA). You could be doing RNA-seq, and then use qPCR for verification, you may want to start with an mRNA fraction so that you dont sequence any rRNA. If youâre working with DNA- you could be using fragmented DNA or gDNA or plasmid DNA; For either type, you want to do sample QC, with either using a spectrophotometer such as a Nanodrop, and looking at the DNA on gel Decide on PCR method (SYBR or Probe) based on budget/ multiplexing Assay design- for this, we ususally go through and develop one or primers, and this can involve bioinformatics, maybe getting copy of transcriptome/genomic regions Optimize assay amplification sensitivity/ amplification efficiency/ specificity- take multiple assay designs and look at all 3 factors May play with cycler temperature to optimize this and possibly go back and redesign our assay Important factor: EVERYTHING MUST BE OPTIMIZED Any time you change something in this experiment, this can affect the total reaction. If you change MM, change instrument, the ramping time, sample purification, means you may have to optimize the other steps in order to keep a good experiment that you can get the optimal data from.
- From Sam: Looking at gene expression analysis. This is an experiment done in our R&D lab a few years ago. It started with human mesenchymal stem cells, extracted from several different sources. The goal of this experiment is to define the probability of the stem cells to become bone/cartalige cell(osteogenesis), or to become a neuron cell (neurogenesis). We can tell which path the stem cells are going down based upon the gene expression. Followed 1 housekeeping gene in this experiment. When doing gene expression experiments, we typically use a housekeeping/control gene. Gene that will not change expression value throughout all data we analyze. It sets the baseline and normalize for all possible changes that could occur. Screened multiple genes to be potential housekeeping genes. Can pick 1 or more to stand as the reference gene. GOI (gene of interest) in this case is TNFa (Tumor necrosis factor alpha). First, going to plate our cells, then add differentiation media Then, take samples at the âzeroâ time point (beginning of experiment), and time point 1 (25% time point), time point 2 (50%), time point 3 (75%), time point 4 (90%) Then, the last time point taken will be when the final cells are completed (16 days or 72 hours) In order to calculate the statistic and reproducibility of the experiment, we will need to have biological replicates. Biological Replicates are extracting the hMSC 3 times and going through the differentiation protocols with these samples. Once you have gone through the qPCR assay and know that it works well, no reason to repeat same cDNA and assay (technical level) Biggest problems will be at the biological level, differentiation medias are variable, so need biological replicates Using at least 3, you can calculate a standard deviation. Final values are going to be: Fold change plus/minus p value Can also calculate confidence interval Fold change values based on saying you have a 2-or 4-fold up-regulated gene, and a statistic that says that it is highly reproducible
- Here is the workflow for this particular experiment, and you can see it is set up how the workflow example looks. We isolate our total RNA, perform our QC measures, reverse transcribe our RNA into cDNA, we prepared an assay design using SYBR Green, and optimized our reaction and ran it on our qPCR machine. Then we did our data analysis.
- So after looking at our example experiment, letâs go over what factors are critical for a successful qPCR assay. You need a good quality template and this is dependent on your sample preparation. If you know youâre going to have a difficult sample and youâre going to have inhibitors, then you can plan ahead for this. If you are working with blood, and you know you are going to use a heparin coated tube, then you can plan to purify out and digest the heparin. If you know youâre going to be working with a stool sample, then You need to undergo a reverse transcription event to convert your RNA to cDNA. To decide on the type of kit youâre going to use, you need to decide what type of reverse transcription reaction you want to do. If you are starting with RNA with a poly A tail, you will want to use a primer that is 3â biased. If you have a fragmented RNA sample, then you want to use primer that uses random hexamers. You can also use both, so that when you do primer design you can design it to the best part of the gene. You also want to include a reverse transcription control to monitor this step and make sure it is working correctly in all of our samples. You can incorporate an artificial RNA and use this to measure the reverse transcription efficiency. This way, if we dont see gene expression in our samples, we know that this is an accurate result and not a failure of our reverse transcription reaction. So if your reaction is 100% efficient, then it turns 1 copy of RNA into cDNA, and if itâs only 10%, then only a 10th of that is copied. If you start diluting and aliquoting that cDNA sample into your qPCR reactions and only have a gene with a few copies available, then you may not be able to detect this. Assay design is important and you want it to be a validated assay. You want the assay you use in your research to be reliable and that it wonât change performance throughout your study, especially if you plan to use this in any future research and possibly to form a diagnostics tool. Our Mastermix will have all of our reagents: the buffers, dNTPs, polymerase, Magnesium, probes and primers, and any additives to control for any high GC content. You should validate any of the assays you design with the same mastermix, and if you plan to change the mastermix then you should go back and revalidate any of your assays. For data analysis, you can build an excel spreadsheet and there are other forms out there. I will briefly go over the streamlined QIAGEN data analysis tool, which lets you upload your Ct values and it calculates your fold change for you.
- Extract total RNA from 18 bases to highest mole weight, archive RNA for future experiments There are several ways to isolate RNA: Qiazol- organic extraction is based on phenol-guanididine based lysis. This setup puts an immediate end to all the Rnases and biological activity, so it stops the sample in time. It can be used for a lot of different cell types and it can remove a lot of the organic inhibitors in your samples. Column based- includes a lysis buffer, it is a lot safer to use and you donât have to worry as much about disposal and waste removal. You do have to be careful about the cell types you use and incorporate a manual disruption of the cells. Both- The advantage of using this is that you can put your sample through an organic extraction, then run it through a column clean-up step, plus you can suspend it in a buffer you can use as long-term storage. This is usually the way to go if you plan to do any experiments involving miRNA. 1. Organic extraction if you want, then precipitate with alcohol and salt step 2. Column based 3. Both methods: difficult cells are difficult to lyse, use organic extraction to lyse cells (instant inactivation of Rnases there; instant end of bio activity, snapshot in time as soon as organic extraction) column-clean-up: good molecular bio grade RNA to store in freezer RNA-seq- might need to exclude RNA clean-up to exclude pRNA or remove rRNA to do deep sequencing
- QC Step: So now that weâve purified our sample, go through the QC step. Letâs talk about qualifying it to check for the purity and integrity of the RNA. If youâre checking for RNA purity and quantity, you can do this using a UV spectrophotometer, such as a nanodrop. The readings you want to take will be at around 260/280 and 260/230. RNA is at maximum absorption at 260nm, so an OD260 reading will give you the amount of nucleic acid you, and an absorbance reading of 1.0 at 260 nm in a to an RNA concentration of about 40 Îźg/ml. The ratios for your 260/280 reading and your 260/230 reading are indicative of whether you have any contaminants in your sample. So you usually want a reading of 1.8-2 or 1.7, depending on which you are measuring. Any lower ratio may mean that you have protiens, Qiazol or Trizol, Carbohdrates in your samples, and you may have to go back and send your sample through an additional cleanup step. So remember that this step does not give any information on the integrity of your sample, so you donât know if this is fragmented or intact. If you are looking at the RNA quality, you can use a denaturing agarose gel or a bioanalyzer. With an agarose gel, you want to run your RNA sample and look at the ribosomal bands. You should get 2 sharp bands and these bands should present for both the 18S and 28S ribosomal RNAs. Some of the newer technologies for this are the bioanalyzer and the QIAxcel system, and the system runs the RNA through a gel matrix and you get an RNA Intergrity Number or RIN number out of this, which is calculated as a ratio of the ribosomal bands. A good RIN is about 7, but this also depends on your experiment, so what buffers and reagents are you using, what reverse transcription are you doing, and depending on these factors your RIN number can vary from being lower of higher. You can see our 2 peaks here which represent our ribosomal bands, and which makes up about 90% of your total RNA, and down here you can see your miRNA band. Again, if you find that you have fragmented DNA, you can design your primer assays to deal with this or you can rethink your isolation method.
- Once we have decided on our isolation method and we know we have sample we can use for our experiment, then we want to work on our reaction components. So how much template do we need to use in our final reaction? 10-1000 copies of NA in per individual qPCR assay (low expressed gene, need 10ng per individual reaction) start with 100pg to 1 ug total RNA per test B. We also want to think about what kind of reaction do we want to do, and this is either a 1-step or a 2-step reaction, and there are pros and cons of doing both methods. In a 1-step reaction, you are performing the reverse transcription reaction and the PCR in same tube. Because of the different enzyme tempertures, you can keep your qPCR from starting prematurely using a Hot-start Taq, which has an antibody that keeps it from working, and put all the reagents in the same tube, run your RT reaction, heat up and activate Taq polymerase to start qPCR reaction. This is usually done if you have a high-throughput laboratory or youâre working with microbial detection, or if youâre working with a smaller amount of template RNA. In a 2-Step reaction, your reverse transcription and your PCR steps are done in separate tubes, and you can do this if your RNA sample is more abundant and less limiting for you. 2-step qPCR is advantageous because of the differences in reagent volumes and you can optimize each reaction. Typically, the magnesium needed for the reverse transcription reaction is around 6-8mm, and for the PCR reaction uses around 1-1.5mm. Higher Mg drives the reverse transcription reaction, and lower amounts give you increased primer specificity in your PCR. C. Read list ( passive reference dye list, such as ROX, etc, depends on machine)
- For our reverse transcription reaction, we want to talk about how we prime the reaction and the type of reverse transcriptase we want to use. We can use a reverse transcriptase with or without an RNase H activity, which allows the RT to degrade RNA in a DNA/RNA hybrid, so you only get 1 copy from that RNA, but may also slow down reactivity. To prime the reaction, you can use Oligo-dT primers or random hexamers or pentamers, or you can use both. You can also use a control RNA using an artificial RNA to measure the reverse transcription efficiency for all samples, and this way you can know that if you donât see any gene expression that this is due to the biology and that this gene is simply expressed below your limit of detection, and not that the RT reaction was ineffective. Some important things to remember are that the RT reaction is linear across all samples, meaning you get 1 copy of cDNA from 1 copy of RNA, so unlike PCR you arenât amplifying anything. Also, more RNA does not mean better results and will not have a large effect on PCR values, and it could possibly inhibit the reaction. The sensitivity of your qPCR experiment is also dependent on whether you have a successful RT reaction, so again it is good to be able to use the artificial RNA mentioned so that you can monitor your efficiency.
- We are ready to amplify our cDNA. Lets explain how PCR works; List components briefly (Polymerase, primers, dNTPs) Polymerase- âthermostableâ i.e. can withstand temperatures Up to ~95C
- Run PCR, thermocycler temp begins to change
- The first step is heating to denature the double-stranded DNA/cDNA and make it single-stranded
- Then the temperature cools and primers start to bind to DNA (TM minus 5 is usually good starting point for annealing temp), 60 degress is good start
- Polymerase will bind...
- ...recognize 3â hyrdoxyl end and extend along the template...
- ...And then finish reaction...
- ... and repeat over and over again...
- Now to explain how to make PCR into a qPCR reaction. Will need to measure the amount of PCR product, either during extension or at the end of the cycle reaction. Measure fluorescence and map fluorescence as a function of the cycle number
- So we talked about what actually goes on in your reaction tube or well and in order to make this reaction a real time PCR reaction, we want to monitor the fluorescence, and there are 2 common ways to do this in qPCR, with SYBR I dye and Taqman Probes. First, we have the DNA intercalating dyes, such as SYBR Green dyes. So, these dyes intercalate or insert themselves into double-stranded DNA, and once they do this the fluorescence increases. This type of detection is used about 50% of the time. This tends to be the most affordable option. SYBR (dsDNA intercalating dyes) When they intercalate, the fluorescnece increases More than 50% uses this Affordable Hydrolysis probes Specific for your amplicon As the DNA polymerase moves through DNA it has 5â to 3â exonuclease activity and cleaves the probes TaqMan probes (from ABI) are common examples of this Pricier option Hybridization probes, have a fluoresence moiety on one end and a quencher on the other end with a stem loop structure, and once the DNA is amplified, the distance between them increases and the fluorescence increases.
- For DNA intercalting dyes, the SYBR Green does not intercalate into SS DNA, only DS DNA. Once a product is created in your tube and DS DNA is available, the dye intercaltes into the DNA and fluorescence increases. high specificity is important with these dyes because the dye cannot distinguish between one DS DNA and another. So that could create: Primer dimers, where the primers will anneal and extend each other, and you can check this from the melting curve Off-target amplification, when the primers recognize multiple genes and you get 2 products made at the same time Amplification of gDNA ALL OF THIS will complicate SYBR Green and give bad data at the end of experiment Make sure that you have: 1 amplicon for each individual target high specificity Advantages: Intercalating dyes are inexpensive and can be used for all of your qPCR experiments One of the disadvantages is that you cannot do multiplexing with the DNA intercalating dyes like you can with the probe assays.
- This is what a typical kinetic curve, or real-time PCR plot, looks like On the Y-axis we have our fluorescent signal, and on the X-axis we have our cycle-number. You can see we have the lower detection value, the linear value, and the plateau phase where we ran out of some reagent In this reaction, we are looking at n=3 replicates for 3 concentrations of a template, and we are looking at 107 copy, 106 copy, and 105 copy Where we have the most product we see amplification first, we can already see amplification at cycle 11. Our Black line is the âfinish lineâ or threshold line in our experiment You can set the threshold line where you see the early amplification, or in the linear area, but you donât want it positioned in the plateau area where the kinetics of the experiment have started to change and you are in a limiting step in the process. For our Final data, we want to use information where the amplification curve intersects the threshold line, and take the cycle number on x-axis as the data point and that is our Ct value. With this, we can use mathematics to determine how much is there, and the amount of fluorescence is proportional to the amount of Double-stranded DNA that we see amplified. You can see we have a 3.3 Ct difference, which is what we would predict to happen.
- Probes work differently than our intercalating dyes, they have a Dual-labeled probe on each end, with a 3â end- quencher and a 5â end- fluorachrom When fluorochrom and quencher are close there is typically no fluorescence As we get polymerization from primers, polymerase comes into contact with probe, it cleaves probe and increases distance of fluorophore from the quencher and you get increased fluorescence This is a Great way to do multi-plexing Multiple reactions in same tube and you can measure all the genes you want at one time Disadvantage: cost and time Each amplicon needs sequence-specific probe Can be $100 or more per probe, so this is costly if you want to look at a lot of genes Typically, start with SYBR green to verify primers are working, then go back in and add hyrdolysis probes
- Same as using the DNA intercalating dye Only difference is the shift of amplification from the left to the right, this is determined by the amount of fluorescence you get and that can be Instrument-specific. BUT the difference between these distances should be the same. The final fold change measurement should be the same, whether with a SYBR assay or a probe assay If you see a linear curve, you may have an off-target amplification that you cant see or primers are not working
- Amplification efficiency: critical; assume 100% when setting threshold value; if you know you donât have 100% efficiency you can take that into account during data analysis, but best way to assure you will get high quality data at the end Sensitivity: if you want to know how many copies you can detect down to, easy to achieve with good assay design; can increase cDNA at beginning if low sensitivity Specificity: make sure you are only amplifying the gene of interest; 1 assay, 1 target; off-target amplification can use-up reagents and can limit reaction even though off-target amplification is not measured ex. If you have 35 cycles Dynamic Range: ability to detect genes with varied expression levels 10 to 109 copies is ideal Reproducibility: Confidence in the accuracy of your results, enables profiling of multiple genes in the same sample All lab members get the same results Technical reproducibility ensures changes seen in results are due to the biology and not the technology itself or sample handling
- To look at the Amplification analysis; 2 ways to calculate: The first is the Standard curve method : this involves diluting a template out, so starting with artificial DNA so we know the copy number, make a dilution series from 1 copy to 1^10 copy. Run qPCR and graph the corresponding threshold value. Calculate using equation 10^(-1/slope of the curve) minus 1*100. Amp efficiency of 100 plus/minus 10 percent is good. Dependent on pipetting ability B. With Single-Curve Analysis you can look at the fluorescence generated and use PCR Miner or DART and input/export numbers Also use this to see the dynamic range using serial dilutions of known template
- Sensitivity: How many copies can I detect? So when you think about sensitivity you want to know an absolute copy number. Method 1: use primers for qPCR reaction to amplify a product, then clone into a vector using T/A cloning, purify the plasmid and calculate how many copies based on amount of DNA since you know the size of vector. Method 2: you can use gDNA as a template and use mass of the gDNA, the number of bases, and assume 1 target per genome. First figure: Different amounts of Human XpressRef⢠Universal Total RNA (pooled from more than 20 different human cell lines) were characterized using Human Inflammatory Cytokines & Receptors RT2 Profiler PCR Arrays. The percentage of detectable genes (those yielding threshold cycle values less than 35) was calculated and plotted against each RNA amount. As little as 25 ng total RNA yields greater than 80 percent positive calls âeven for cytokine genes in un-induced cells
- You want to run a disassociation curve at the end of the reaction, and you can also take it a step further and run it in a gel. For the curve, youâre looking at a single curve at the expected TM.
- As it melts from double stranded to single stranded, we see a large drop in the fluorescence.
- What the machine does is it takes the negative 1st derivative , which shows us to look at the rate of change and that will be the peak of our curve. If you see a peak here at lower temperatures, that is usually a primer dimer. If you see 2 peaks here, this isnât always bad and could be a splicing event, so you may want to go back and do some sort of sequencing to look ta this.
- Data analysis is dependent on having replicates. Our biological replicates show us the difference based on the biology. The biological replicates tend to have the most biological variability. Once you have a validated assay and you can see about 20 copies or more, you dont need to do technical replicates unless you want to monitor that. You want to have at least 3 biological replicates so you can caluclate a standard deviation. If your validating your assays, youll want to do your techincal replicates.
- What your looking at is the fluorescence and you want to set the baseline. Most of the instruments today will have something to auto set the baseline by looking at the rate of change, or you can also go through and set your baseline by starting at cycle 2, look for the earliest amplification, and then move back about 2 cycles
- Now you need to set your threshold line to determine the Ct values, and you can do this by looking at the log view. You can see the y axis is now the log value. You can see the background noise here and the rate limiting step, and teh log linear step here. You want to go through and see where all of your curves are parallel to each other and set your threshold here. You want to look at the ratio of these Ct values and calculate your fold change.
- The housekeeping gene is a gene that isnât going to change itâs expression levels throughout you experiment. The reference gene is aimed to normalize any variation in the following: Sample prep & handling (e.g use the same number of cells from a start) RNA isolation (RNA quality and quantity) Reverse transcription efficiency across samples/experiments PCR reaction set up PCR reaction amplification efficiencies
- Here is a list of our popular housekeeping genes. Ribosomal isn't ideal because it isnât representative of low-expressed genes. You may want to look for structural genes or metabolic genes. You can get this list as well after the webinar, I will send out the slides to everyone. You can also look at several housekeeping genes, the relative expression levels might be important, because if you have a low-expressing housekeeping gene, you know something could be wrong because these are likely important for cell survival.
- Skip to data analysis webpage
- Skip to data analysis webpage
- Skip to data analysis webpage
- Skip to data analysis webpage
- All of our calculations can be greatly simplified if you have some sort of data analysis module. You can upload your data, and it can calculate some statistics if you have your 3 biological replicates, give you housekeeping gene recommendations, take you through some of the analysis steps.
- So with our RT2 profiler system, we will look at amplifying a large number of genes that are related in some way by pathway or disease, once we understand whats going on with a qPCR experiment. HKG- GDC- this is a positive call for any gDNA contamination, and itâs a multi-gene assay so we can usually detect any contamination RTC- This allows us to detect our artificial RNA, and this is a way we can monitor the RT across a large amount of samples, and we can see any technical issues PPC- this is a way to monitor that the instrument and the optics are working, the mastermix is working. We can also use teh PPC to set a universal threshold value. There are also propriety controls
- Water to use for dilutions: needs to be NA-free,