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Dr. Chaim Wachtel April 11, 2013 Introduction to Real-Time PCR.

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Presentation on theme: "Dr. Chaim Wachtel April 11, 2013 Introduction to Real-Time PCR."— Presentation transcript:

1 Dr. Chaim Wachtel April 11, 2013 Introduction to Real-Time PCR

2 Real-Time PCR What is it? How does it work How do you properly perform an experiment Analysis

3 Michael Smith The Nobel Prize in Chemistry 1993 was awarded "for contributions to the developments of methods within DNA-based chemistry" jointly with one half to Kary B. Mullis "for his invention of the polymerase chain reaction (PCR) method"and with one half to Michael Smith "for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies".

4 PCR – A simple idea Polymerase Chain Reaction: Kary Mullis (1983) In vitro method for enzymatically synthesizing DNA The reaction uses two oligonucleotide primers that hybridize to opposite strands and flank the target DNA sequence that is to be amplified A repetitive series of cycles gives exponential accumulation of a specific DNA fragment –Template denaturation –Primer annealing –Extension of annealed primers by the polymerase The number of target DNA copies doubles every PCR cycle (20 cycles  2 20 ≈10 6 copies of target)

5 Principle of PCR

6 Difference PCR vs real-time PCR? Fluorescence is measured every cycle (signal  amount of PCR product). Curves rise after a number of cycles that is proportional to the initial amount of DNA template. Comparison with standard curve gives quantification.

7 Real-Time and End Point End point Real time

8 MIQE: the minimum information for the publication of qPCR experiments. http://www.rdml.org/miqe.php

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12 The mRNA of the Arabidopsis Gene FT Moves from Leaf to Shoot Apex and Induces Flowering Tao Huang, Henrik Böhlenius, Sven Eriksson, François Parcy, and Ove Nilsson Science 9 September 2005: 1694-1696. 2005: Signaling Breakthroughs of the Year

13 Retraction WE WISH TO RETRACT OUR RESEARCH ARTICLE “THE MRNA OF THE ARABIDOPSIS GENE FT MOVES from leaf to shoot apex and induces flowering” (1). After the first author (T.H.) left the Umeå Plant Science Centre for another position, analysis of his original data revealed several anomalies. It is apparent from these files that data from the real-time RT-PCR were analyzed incorrectly. Certain data points were removed, while other data points were given increased weight in the statistical analysis. When all the primary real-time RT-PCR data are subjected to correct statistical analysis, most of the reported significant differences between time points disappear. Because of this, we are retracting the paper in its entirety.

14 Real-Time Machines How do they work What can you do with one –Gene expression –SNP detection –DNA detection (quantify) How do you use them –Experiment design Everything you need to know and more about RNA and RT-PCR

15 “Fifty Years of Molecular Diagnostics” Clin Chem. 2005 Mar;51(3):661-71 (C.Wittwer, ed.) First real-time PCR, 1991 PCR tube in thermocycler spectrofluorometer fiberoptic

16 First commercial real-time PCR instruments ABI 7700 – laser/fiberoptic-based ABI 5700 – CCD camera-based Idaho Technology LightCycler – capillary tubes

17 7900HT Fast Real-Time PCR System (Sol Efroni’s lab) RT-PCR machines at Bar Ilan AB StepOnePlus Fast Real-Time PCR System Qiagen’s Rotor-gene (Oren Levy’s lab) Bio-Rad CFX-96 Thermo PikoReal (Bachelet Lab)

18 Rotor-gene

19 Probing alternatives Non-specific detection Dyes: SYBR Green I, BEBO, BOXTO, EvaGreen... Specific detection TaqMan probe Molecular Beacon Light-Up probe Hybridization probes Primer based detection Scorpion primers QZyme Lux primers

20 SYBR Green binds to dsDNA SYBR Green binds to DNA, particularly to double-stranded DNA, giving strongly enhanced fluorescence. SYBR Green is sequence- dependent!

21 The TaqMan Probe The TaqMan probe binds to ssDNA at a combined annealing and elongation step. It is degraded by the polymerase, which releases the dye from the quencher.

22 Multiplex Q-PCR Detection of two (or more) different target sequences in the same reaction.

23 qPCR technical workflow Sampling DNA Extraction RNA Extraction DNase treatment Reverse Transcription qPCR Data Analysis

24 Nucleic acid isolation and purification

25 Overview Sampling Accessibility and lysis Commonly used techniques RNA considerations Quality control

26 Why sample preparation? Make target available Remove inhibitors Remove fluorescent contaminants Preserve target integrity Concentrate target

27 Path Genomic DNA Plasmid DNA mRNA Total RNA Fragment DNA Nuclear RNA Phage DNA ReverseTranscription Real-time PCR DNARNA Purification IsolationDisruption

28 Accessibility Sample disruption and homogenization –Mechanical Grinding, Sonication, Vortexing, Polytron –Physical Freezing –Enzymatic Proteinase K, Lysozyme, Collagenase –Chemical Guanidinium isothiocyanate (GITC), Alkali treatment, CTAB

29 Lysis –Complete or partial lysis? –Chaotropic lysis buffers: SDS, GITC, LiCl, phenol, sarcosyl –Gentle lysis buffers: NP-40, Triton X-100, Tween, DTT

30 Purification principles Characteristics of nucleic acids –Long, unbranched, negatively charged polymers Examples: –Differential solubility –Precipitation –Strong affinity to surface Factors: –pH, [salt], hydrophobicity

31 Purification techniques Solution based- eg Tri reagent, CsCl gradient Precipitation- ethanol, needs salt, multiple factors can influence precipitation Membrane based- spin columns (Qiagen and the like) Magnetic bead based

32 Solution based isolation Most methods use hazardous reagents Phenol/Chloroform extraction –Proteins, lipids, polysaccharides go into the organic phase or in the interphase. –DNA/RNA remains in aqueous phase Caesium chloride density gradient ultracentrifugation –Time consuming Acid guanidine phenol chloroform extraction –Commonly called TRIzol

33 Precipitation purification Nucleic acids precipitate in alcohols Salt (NaCl, NaAc) facilitates the process Important factors: Temperature, time, pH, and amount

34 Membrane based isolation Anion exchange technology Spin column / silica gel membrane –Chaotropic salts (e.g. NaI or guanidine hydrochloride) bind H 2 O molecules –Loss of water from DNA changes shape and charge –DNA binds reversibly to silica membrane

35 Purification – GITC vs. column Organic liquids Pro: –Higher yield –Can handle larger amounts of cells –Better for troublesome tissues (fatty tissue, bone etc) Con: –Higher DNA contamination (for RNA isolation) –Separate DNase I digestion with additional purification Spin columns Pro: –Less contaminating DNA (for RNA isolation) –On column DNase digestion Less loss of RNA –Higher quality –Easy to use Con: –Limited loading capacity –More expensive (?)

36 RNA Considerations RNA is chemically and biologically less stable than DNA Extrinsic and intrinsic ribonucleases (RNases) –Specific and Nonspecific inhibitors

37 Stabilizing conditions Work on ice Process immediately Flash freeze sample in liquid nitrogen and store at -70°C until later use Store samples in stabilization buffer

38 Storage of nucleic acids Nuclease-free plasticware Eluted in nuclease-free water, TE or sodium citrate solution RNA: Neutral pH to avoid degradation Aliquot sample to avoid multiple freeze-thaw cycles Isolated RNA should be stored at -20 deg C or -70 deg C for even better protection in ethanol and not water.

39 Quality Control Spectroscopic methods –Concentration, [NA] = A260 x e mg/ml –Purity: A260 / A280 (≈1.8 for DNA, 2.0 for RNA) Dyes –Quantification by fluorescence of DNA/RNA- binding dyes (Qubit) Electrophoresis (28S and 18S bands)

40 What is the BioAnalizer? Microfluidic separations technology RNA - DNA - Protein 1µl of RNA sample (100 pg to 500 ng) 12 samples analyzed in 30 min Integrated analysis software: –Quantitation –Integrity of RNA

41 Bioanalyzer

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45 RNA Integrity: RQI Good RNA Quality Bad RNA Quality 10RNA Quality Indicator1

46 Publications on RNA integrity

47 DNase I treatment of RNA samples No RT, DNase RT, DNase RT, No DNase No RT, No DNase

48 qPCR technical workflow Sampling DNA Extraction RNA Extraction DNase treatment Reverse Transcription qPCR Data Analysis

49 Reverse transcription RT

50 Outline Priming efficiency Reproducibility Properties of Reverse transcriptase RNA concentrations

51 General description of RT reaction Reverse Transcriptases are RNA- dependent* DNA polymerases that catalyze first strand DNA synthesis in presence of a suitable primer+ as long as it has a free 3’ OH end. *Can use also single strand DNA as template. + Can be either RNA or DNA.

52 RT priming

53 RT with Gene-Specific Priming

54 RT with Oligo(dT) Priming

55 RT with Random Hexamer Priming

56 Real-time PCR using different RT primers

57 Real-time PCR with different RT primers

58 Dependence on priming strategy

59 Dependance of priming method  -tubulinCaVIDGAPDH Insulin IIGlut 2 hexamers19,526,515,816,927,5 oligo dT18,128,816,615,928,4 GSP18,828,716,417,431,8 mix19,127,916,316,629,3 max  Ct1,42,30,81,54,4 Gene RT priming method

60 Specificity of specific priming  -tubulinCaVIDGAPDH Insulin IIGlut 2  -tubulin18,828,71918,830,6 CaVID2718,719,922,8- GAPDH23,430,116,420,129,7 Insulin II23,531,62017,431 Glut 225,831,922,7 31,8 no RT primer27,633,723,623,132,6 NTC ~ 35 PCR primers used RT primers used

61 37ºC GAPDH 3’ 60ºC 18 unpaired bases 24 unpaired bases Algorithm: mfold

62 Comparison of reverse transcriptases Temp Ref: Ståhlberg et al. Comparison of reverse transcriptases in gene expression analysis. Clin.Chem. 50(9); 1678-1680 (2004) MMLV RNase H- Minus (Promega, Germany)37 M-MLV (Promega)45 Avian Myeloblastosis Virus (AMV) (Promega) 37 Improm-II (Promega)45 Omniscript (Qiagen, Germany)37 cloned AMV (cAMV) (Invitrogen, Germany)45 ThermoScript RNase H- (Invitrogen) 50 SuperScript III RNase H- (Invitrogen)50

63 100 – fold variation in RT yield

64 8 transcriptases tested on 6 genes

65 Experimental design to study linearity

66 Effect of carrier + tRNA - tRNA

67 Effect of carrier + tRNA - tRNA

68 RNA dilutions Water Yeast tRNA Oligo(dT)Random Hexamers

69 Conclusions The RT reaction shows higher technical variability than QPCR There is no optimum priming strategy Gene specific primers must target accessible regions The RT yield changes over 100-fold with the choice of reverse transcriptase The yield variation is gene specific RT yield is proportional to the amount of template in presence of proper carrier Typical RT yield is 10-50 % RT-QPCR is highly reproducible as long as the same protocol and reaction conditions are used The efficiency of the RT reaction varies from gene to gene and depends on the conditions – run the RT of all samples using exactly the same protocol and reagents under the same conditions


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