بسم الله الرحمن الرحیم

بخش مهندسی بیوتکنولوژی کشاورزی آموزش مبانی Real time PCR بخش مهندسی بیوتکنولوژی کشاورزی

History The routine study of DNA became practical with the invention of the polymerase chain reaction (PCR) by Kary Mullis in 1983. Despite advances in PCR, quantitation of DNA or RNA in cells remained a difficult task until 1993 when Russell Higuchi and collegues introduced real-time. Higuchi R, Fockler C, Dollinger G, Watson R (1993) Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions. Biotechnology 11(9): 1026–1030.

Real Time PCR Application Gene expression Microarray and RNA seq validation DNA methylation(QMSP, MSRE-PCR, MS-HRM ) SNP Genotyping & Alleilic discrimination Copy Number Detection(CNV) Pathogen detection and quantification

What exactly is real-time quantitative PCR? Real-time PCR is the continuous collection of fluorescent signal from one or more polymerase chain reactions over a range of cycles. Quantitative real-time PCR is the conversion of the fluorescent signals from each reaction into a numerical value for each sample.

Real-time PCR Principles based on the detection and quantitation of a fluorescent reporter In stead of measuring the endpoint we focus on the first significant increase in the amount of PCR product. The time of the increase correlates inversely to the initial amount of DNA template

Real-time PCR advantages * not influenced by non-specific amplification * amplification can be monitored real-time * no post-PCR processing of products *~ high throughput * Broad dynamic range * requirement less RNA than conventional assays * most specific, sensitive and reproducible

Real-time PCR vs End Point Detection

Polymerase Chain Reaction (PCR) PCR reaction components: Tempelate Primers dNTP mix MgCl2 Thermostable DNA polymerase Buffer

Quantification in PCR To understand real-time PCR, let’s imagine ourselves in a PCR reaction tube at cycle number 25… What’s in our tube, at cycle number 25? A soup of nucleotides, primers, template, amplicons, enzyme, etc. 1,000,000 copies of the amplicon right now.

Quantification in PCR What was it like last cycle, 24? Almost exactly the same, except there were only 500,000 copies of the amplicon. And the cycle before that, 23? Almost the same, but only 250,000 copies of the amplicon. And what about cycle 22? Not a whole lot different. 125,000 copies of the amplicon.

Quantification in PCR ? So, right now we’re at cycle 25 in a soup with 1,000,000 copies of the target. What’s it going to be like after the next cycle, in cycle 26?  

Quantification in PCR Realistically, at the chain reaction progresses, it gets exponentially harder to find primers, and nucleotides. And the polymerase is wearing out. So exponential growth does not go on forever!

Quantification in PCR Let’s imagine that you start with four times as much DNA as I do…picture our two tubes at cycle 25 and work backwards a few cycles. Cycle 25 Cycle Me You 23 250,000 1,000,000 24 500,000 2,000,000 25 4,000,000

Quantification in PCR So, if YOU started with FOUR times as much DNA template as I did… …Then you’d reach 1,000,000 copies exactly TWO cycles earlier than I would!

Quantification in PCR What if YOU started with EIGHT times LESS DNA template than I did? Cycle Me You 25 1,000,000 125,000 26 2,000,000 250,000 27 4,000,000 500,000 28 8,000,000 Cycle 25

Quantification in PCR What if YOU started with EIGHT times LESS DNA template than I did? You’d only have 125,000 copies right now at cycle 25… And you’d reach 1,000,000 copies exactly THREE cycles later than I would!

Amplification plot

Amplification plot

Amplification plot

Exponential phase Linear ~20 to ~1500

Exponential phase Linear ~20 to ~1500

Threshold Cycle (CT)   21 23 26

Serial Dilution SERIES OF 10-FOLD DILUTIONS

Efficiency calculation threshold Ct

Efficiency calculation  

Optical Detection Systems www.biorad.com 2a. excitation filters 2b. emission filters 1. halogen tungsten lamp 4. sample plate 3. intensifier 5. ccd detector 350,000 pixels Chapter1 from rna methodology book

Fluorescence Chemistry DNA binding agents SYBR Green, Eva Green, BOXTO, LCGreen®, SYTO® 9 and BEBO Hydrolysis probes TaqMan hybridization probes FRET

Fluorescence resonance energy transfer FRET is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance FRET depends on the donor and acceptor molecules being in close proximity (10– 100 A)

DNA binding agents SYBR® Green I advantages Low cost assay Easy design and set up Useful for generating melt curves and HRM analysis SYBR® Green I disadvantages Non specific system Not adapted to multiplex

Apply Excitation Wavelength 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Extension Taq ID ID 5’ 3’ 5’ 5’ ID ID ID Taq 5’ 3’ Apply Excitation Wavelength l Taq ID 5’ 3’ ID ID 5’ 5’ ID ID Taq 3’ l

Hydrolysis probes Double-Dye probes advantages Widely used, several modifications possible Multiplex capabilities Double-Dye probes disadvantages More expensive than SYBR® Green I Problem in Design

TaqMan® probe types TaqMan probe TaqMan MGB probe

Molecular Beacon

hybridization probes

Amplification Plots & Basic Terms Rn: Fluorescence signal normalized with passive reference dye signal (Fluorescence/dye signal) Passive Reference Dye: An internal reference to correct well to well non‐PCR fluctuations. ROX is the most commonly used reference dye. ΔRn: Fluorescence signal with baseline subtracted (background signal subtracted) Baseline: background noise level before a significant amplification occurs (3‐15 cycles)

Assay Validation Specificity Analysis Precision and Variability Melt Curves Amplicon Size Analysis Sequencing Precision and Variability

The hallmarks of an optimized qPCR assay Efficiency:amount of PCR product dubles during each cycle of exponential amplification Specificity:target DNA is the only reaction product Sensetivity:broad linear range that encompases the entire range of templet concentration Reproducibility:variability across assay replicates is minimal

Determining Gene Expression Changes

Why study RNA? All cell and tissue functions are ultimately governed by gene expression Fundamental themes in RNA studies: Abundance Structural Cell-free in vitro translation Synthesis of cDNA Chapter1 from rna methodology book

Types of RNA rRNA(most abundant) mRNA(most diverse) tRNA Different class of Small RNAs Chapter1 from rna methodology book

Gene expression methods Northern blot Nuclease protection SAGE SSH cDNA AFLP Micro array Semi quantative PCR Real time PCR Digital PCR RNA-seq

Pipeline for gene expression analysis by Real time PCR RNA extraction RNA qualification and quantification cDNA synthesis PCR reaction

RNA qualification and quantification UV spectrophotometry Microfluidic analysis (2100 Bioanalyzer, Experion,qiaexcel ) RNA binding dyes(RiboGreen) Qualification Agarose Gel

cDNA synthesis

cDNA synthesis There are three ways to prime a reverse transcriptase reaction: Oligo-dT Random primers Assay-specific primers

DNA Polymerase The heart of the modern PCR is the addition of a thermostable DNA polymerase Hot start by: Antibody-mediated(1min 94ºC) Chemical-mediated (10min 94ºC) Organism Thermus aquaticus MW 94KDa Number of amino acids 832 Single chain or subunits single Extension rate 2-4 Kb/min Half life @ 95ºC 40 min Processivity 50-60 bases 5’–3’ exonuclease activity yes

One-step and Two-step RT-PCR Procedure Advantages Two-step RT-PCR Multiple PCRs from a single RT reaction Flexibility with RT primer choice Enables long-term storage of cDNA One-step RT-PCR Easy handling Fast procedure High reproducibility Low contamination risk

Effect of RT volume added to real time PCR

Primer and probe design Length of PCR product should ideally be less than 250 bp Avoid complementary sequences within and between primers and probes Avoid mismatches Avoid a 3'-end T as this has a greater tolerance of mismatch Length: 18–28 nucleotides GC content: 40–60% The probe melting temperature in general should be ~10ºC higher than the forward or reverse primer. Do not put G at the 5’ end of the probe as this will quench reporter fluorescence

Primer and probe design Tools Primer3 (http://www.genome.wi.mit.edu/cgi- bin/primer/primer3_www.cgi) Idt(www.idtdna.com) Beacon designer Primer Express® (http://www.appliedbiosystems.com). Premier Biosoft International (http://www.premierbiosoft.com).

Quantification strategies Absolute quantification Standard curve method Relative quantification Standard curve method With PCR efficiency correction Comparative CT Method Without PCR efficiency correction

Absolute quantification The absolute quantitation assay is used to quantitate unknown samples by interpolating their quantity from a standard curve

Absolute quantification Features of an appropriate standard Primer and probe binding sites identical to the target to be quantified Sequence between primer binding sites identical or highly similar to the target sequence Sequences upstream and downstream from the amplified sequence identical or similar to the“natural” target Equivalent amplification efficiencies of standard and target molecules

Absolute quantification

Housekeeping Genes Genes that are widely expressed in abundance and are usually used as reference genes for normalization in real-time PCR with the assumption of ‘constant expression’. The current trend is first to check which housekeeping genes are suitable for the target cell or tissue and then to use more than one of them in normalization.

Housekeeping Genes A Reference Gene is aimed to normalize possible variations during: 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

Relative quantification by Standard curve method dilutions target DNA dilutions reference DNA target primers reference triplicates cDNA

Relative quantification by Standard curve method ‘copy number’ target gene control Dilution curve target gene ‘copy number’ target gene experimental  

Relative quantification by Standard curve method ‘copy number’ reference gene experimental Dilution curve reference gene ‘copy number’ reference gene control  

Relative quantification by Standard curve method  

Relative quantification Determination of the changes in steady state mRNA levels of a gene across multiple samples and expresses it relative to the levels of another RNA. Calibrator: A single reference sample used as the basis for relative-fold increase in expression studies To determine the level of expression, the differences (Δ) between the threshold cycle (Ct) or crossing points (CP) are measured. Comparative CT Method (ΔΔ CT Method)

Relative quantification gene expression can be relative to: an endogenous control, e.g. a constant expressed reference gene or another GOI a reference gene index, e.g. consisting of multiple averaged endogenous controls

Without PCR efficiency correction  

Without PCR efficiency correction if the PCR efficiency is only 0.9 instead of 1.0, the resulting error at a threshold cycle of 25 will be 261%. The calculated expression level will be 3.6-fold less than the actual value.  

The efficiency corrected calculation method

Example

Example ΔCt _GAPDH = Ct control - Ct treated ΔCt _GAPDH = 14.0 – 13.7   ΔCt _GAPDH = Ct control - Ct treated ΔCt _GAPDH = 14.0 – 13.7 ΔCt _GAPDH = 0.3

Factors affecting the accuracy of Real-Time PCR Pre-analytical steps Tissue sampling and storage, RNA extraction and storage, RNA quantity and quality control Optimized RT and PCR performance specificity, sensitivity, reproducibility, and robustness Post-PCR data procession data acquisition, evaluation, calculation and statistics

Softwares REST Q-Gene qBASE plus DART-PCR linReg PCR