SIBC 512: Real-time PCR. Assistant Professor Chatchawan Srisawat MD. Ph.D

Transcription

1 SIBC 512: Real-time PCR Assistant Professor Chatchawan Srisawat MD. Ph.D

2 REAL-TIME QUANTITATIVE PCR In many applications, accurate quantitation of the number or amount of the interesting gene or its products present in the specimens are desirable. e.g. - Gene expression analysis -> level of gene expression - Diagnostics -> The ability to determine the amount of pathogens or copy number of oncogenes or diseased genes could be used to indicate the disease severity.

3 REAL-TIME QUANTITATIVE PCR Based on the principle of PCR, the amount of PCR products can indirectly reflect the starting copy number of the DNA template if PCR starts with N template copies, the yield will be ~ N2 n. If the PCR amplifcation is done for a fixed cycle number (e.g. 20 or 30) and the PCR products are analyzed on agarose gel, the intensity of the bands generally correlates with the amount of the starting DNA templates. (End point analysis)

4 REAL-TIME QUANTITATIVE PCR Difficulties associated with the end-point analysis included: (i) quantitation must be done when the amplification is within the log phase where the PCR product is directly proportional to the starting copy number. not easy to tell becuase the PCR products are not continously monitored. (ii) The usual method to detect the product once linear amplification was achieved (e.g. electrophoresis, southern blot, etc) is tedious and timecomsuming.

5 PRINCIPLE OF REAL-TIME QUANTITATIVE PCR Real-time PCR or quantitative PCR (qpcr) is a recently developed technique (1996) and has been increasingly popular for PCR product quantitation. It avoid the drawbacks of the previously-mentioned PCR quantiation. a) PCR products are monitored continously (or in real-time) to make sure the amplication is in the log phase. End-point analysis Real-time analysis b) PCR products can be easily detected from the PCR tubes by the real-time PCR machine (not a regular PCR machine). Detection of up to 384 reactions in less than a minute

6 PRINCIPLE OF REAL-TIME QUANTITATIVE PCR SYBR green Pros: simple, no need to design the probes Cons: unable to differentiate signals from non-specific products unable to do multiplex PCR

7 PRINCIPLE OF REAL-TIME QUANTITATIVE PCR Principle of SyBr green.flv

8 PRINCIPLE OF REAL-TIME QUANTITATIVE PCR Fluorescence Energy Resonance Transfer (FRET) Reporter Quencher PCR primer No signals Hydrolysis or Taqman probes 5-3 exonuclease activity of Taq The increase in fluorescence measured cycle by cycle is a direct consequence of the amplification process.

9 PRINCIPLE OF REAL-TIME QUANTITATIVE PCR The signals are detected every cycle by real-time PCR instruments. Stratagene MX3005P.flv

10 PRINCIPLE OF REAL-TIME QUANTITATIVE PCR Stratagene MX3005P.flv

11 PRINCIPLE OF REAL-TIME QUANTITATIVE PCR The signals are detected every cycle by real-time PCR instruments. Real-time PCR instruments enable detection of PCR products in the linear phase. No subsequent detection is required.

12 PRINCIPLE OF REAL-TIME QUANTITATIVE PCR Ct (threshold cycle) within the log phase is determined. X t = X 0 2 Ct log 2 [X t ] = log 2 [X 0 ] + Ct [Ct] = log 2 [X t ] log 2 [X 0 ] [Ct] = K log 2 [X 0 ] X t = number of copies at threshold cycle X 0 = starting tempate (cycle 0)

13 PRINCIPLE OF REAL-TIME QUANTITATIVE PCR [Ct] = K log 2 [X 0 ] [Ct] = K 3.32log[X 0 ] A log plot of Ct and copy number Unknown samples can be quantitated using a standard curve to obtain the number of copies in the sample.

14 ANALYSIS OF REAL-TIME QUANTITATIVE PCR DATA Absolute vs. Relative quantitation Absolute quantitation e.g. number of c-myc mrna 2500 copies/cells, viral load in the blood 400 copies/ml of blood Require standard curve preparation Relative quantitation In some situations, it may be unnecessary to determine the absolute transcript copy number and reporting the relative change will suffice. e.g. - The expression of c-myc is 3.5-fold higher in cancer compared with normal tissues. - The level of COL1A1 mrna in sirna-treated fibroblasts is 1% of that in untreated fibroblasts.

15 ANALYSIS OF REAL-TIME QUANTITATIVE PCR DATA Relative quantitation e.g. - The expression of MYC is 3.5-fold higher in cancer compared with normal tissues. Technical terms to know before getting started. - Gene of interest (GOI) -> MYC - Calibrator = control samples (e.g. normal tissues, cells from Day 0 or untreated cells, control sirna-treated group). The level of expression calibrator is set at 1 (or 100%). -Unknown = experiment or unknown samples (e.g. cancer tissues, cells at various interavals after treatment, Gene-specific sirna) Relative expression of MYC = Level of MYC in unknown Level of MYC in calibrator e.g. Ct of MYC in unknown and calibrator = 25 and 27 cycles

16 ANALYSIS OF REAL-TIME QUANTITATIVE PCR DATA Relative quantitation e.g. - The expression of MYC is 3.5-fold higher in cancer compared with normal tissues. - Generally, the level of GOI is normalized with the level of an endogenous reference gene or normalizing gene, e.g. housekeeping genes, whose expression is relatively constant in various tissues and conditions. - This approach provides a method for correcting results for differing amounts of input RNA. - This is particularly attractive when it is not practical to measure the amount of input RNA by other methods. Such situations include when only limited amounts of RNA are available or when high-throughput processing of many samples is desired. Example of normalizing gene Actin, GAPDH, Tubulin, Aldolase A

17 ANALYSIS OF REAL-TIME QUANTITATIVE PCR DATA Relative quantitation e.g. - The expression of MYC is 3.5-fold higher in cancer compared with normal tissues. If GAPDH is used as a normalizing gene, Relative expression of MYC/GAPDH in calibrator = Level of MYC in calibrator Level of GAPDH in calibrator Relative expression of MYC/GAPDH in unknown = Level of MYC in unknown Level of GAPDH in unknown Eq.1 Eq.2 Relative expression of MYC in unknown compared with calibrator = Eq.2 Eq.1

18 EXPERIMENTAL PROCEDURES See the handout

19 EXPERIMENTAL PROCEDURES Divide into 4 groups ( a group of ~ 4 students) To prepare master mix, Group 1 & 2 each group prepares the assay reactions of beta-actin and GAPDH (e.g. 1A actin, 1B GAPDH). * The students working with real-time PCR experiments must know how to use automatic pipette accurately because real-time quantiation is a sensitive quantitative assay.

20 EXPERIMENTAL PROCEDURES Divide into 4 groups ( a group of ~ 4 students) To prepare master mix, Group 3 & 4 each group prepares the standard curve to determine the amplification efficiency (e.g. 3A- actin, 3B GAPDH)

21 EXPERIMENTAL PROCEDURES Please read the following reference paper before class tomorrow. Analysis of Relative Gene Expression Data Using Real- Time Quantitative PCR and the 2- Ct Method. METHODS 25, (2001) doi: /meth ). Click to download the paper.

22 2 nd session

23 DISCUSSION Analysis of real-time PCR data Gene of interest or target gene = level of Actin Normalizing (reference) gene = GAPDH Unknown or test or experiment group = treated cells at day 3 and 7 Calibrator or control group = untreated cells at day 0 If GAPDH is used as a normalizing gene, Relative expression of Actin/GAPDH in unknown = Level of Actin in unknown Level of GAPDH in unknown Relative expression of Actin/GAPDH in calibrator = Level of Actin in calibrator Level of GAPDH in calibrator Eq.1 Eq.2 Relative expression of Actin in unknown compared with calibrator = Eq.1 Eq.2

24 DISCUSSION Analysis of real-time PCR data Let A, G = copies number of Actin and GAPDH respectively C T = threshold cycle, cb = calibrator, unk = unknown Relative expression of Actin/GAPDH in unknown = Level of Actin in unknown Level of GAPDH in unknown Eq.1 X n = X 0 * 2 n A CT-A,unk = A unk * 2 C T-A,unk G CT-G,unk = G unk * 2 C T-G,unk Relative expression of Actin/GAPDH in unknown = Level of Actin in unknown = A unk Level of GAPDH in unknown G unk

25 DISCUSSION Analysis of real-time PCR data Relative expression of Actin/GAPDH in unknown = Level of Actin in unknown = A unk Level of GAPDH in unknown G unk A unk = A CT-A,unk G unk = G CT-G,unk 2 C T-A,unk 2 C T-G,unk A CT-A,unk A unk = 2 C T-A,unk A CT-A,unk x 2 C T-G,unk G unk G CT-G,unk G CT-G,unk x 2 C T-A,unk 2 C T-G,unk A CT-A,unk x 2 (C T-G,unk - CT-A,unk) G CT-G,unk

26 DISCUSSION Analysis of real-time PCR data Relative expression of Actin/GAPDH in unknown = Level of Actin in unknown = A unk Level of GAPDH in unknown G unk A unk = A CT-A,unk x 2 (C T-G,unk - CT-A,unk) G unk G CT-G,unk A CT-A,unk x 2 - (C T-A,unk - CT-G,unk) G CT-G,unk A CT-A,unk x 2 - C T,unk G CT-G,unk

27 DISCUSSION Analysis of real-time PCR data The same calculation is applied in the case of calibrator. Relative expression of Actin/GAPDH in calibrator = Level of Actin in calibrator = A cb Level of GAPDH in calibrator G cb A cb = A CT-A,cb x 2 - C T,cb G cb G CT-G,cb

28 DISCUSSION Analysis of real-time PCR data Relative expression of Actin/GAPDH in unknown = Level of Actin in unknown Level of GAPDH in unknown Relative expression of Actin/GAPDH in calibrator = Level of Actin in calibrator Level of GAPDH in calibrator Eq.1 Eq.2 Relative expression of Actin in unknown compared with calibrator = Eq.1 Eq.2 A CT-A,unk x 2 - C T,unk A unk G unk = G CT-G,unk A cb A CT-A,cb x 2 - C T,cb G cb G CT-G,cb

29 DISCUSSION Analysis of real-time PCR data C T threshold cycle = the cycle number at which the amount of amplified target reaches a fixed threshold. GAPDH cb actin unk GAPDH unk actin cb GAPDH actin At threshold cycles Level of actin in calibrator at C T Level of GAPDH in calibrator at C T = Level of actin in unknown at C T Level of GAPDH in unknown at C T

30 DISCUSSION Analysis of real-time PCR data A unk A CT-A,unk x 2 - C T,unk G unk = G CT-G,unk A cb A CT-A,cb x 2 - C T,cb G cb G CT-G,cb = 2 - C T,unk 2 - C T,cb = 2 -( C T,unk - CT,cb) = 2 - C T

31 DISCUSSION Analysis of real-time PCR data Relative expression of actin/gapdh in unknown = Level of actin in unknown A cb Level of GAPDH in unknown G cb Relative expression of actin/gapdh in calibrator = Level of actin in calibrator A unk Level of GAPDH in calibrator G 0,unk Eq.1 Eq.2 Relative expression of actin in unknown compared with calibrator = Eq.1 = Eq.2 A unk G unk A cb G cb = 2 - C T CT = -( CT,unk - CT,cb)

32 DISCUSSION Analysis of real-time PCR data Xn = the number of target copies at cycle n of PCR X0 = starting number of target copies. EX = the efficiency of target amplification n = the number of PCR cycles. If the efficiency of PCR amplification of this gene is 100%, the (1+Ex) is 2. X n = X 0 * 2 n

33 DISCUSSION Analysis of real-time PCR data Calculate relative expression of actin in the treated cells at day 3, and day 7 compared with the untreated control cells at day 0. After you finish calculatin, then compare with the results calculated by the software.

34 DISCUSSION Analysis of real-time PCR data Approximation method assume that amplification efficiencies of both genes are equal and close to 100%, which might not be true in some instances. The amplification efficiency can be calculated from the standard curve of serially-diluted samples. MX Pro software can let the researchers to change the amplification efficiencies to get more accurate quantitation.

35 DISCUSSION Analysis of real-time PCR data Tutorial videos Real Time QPCR Data Analysis Tutorial part I.flv Real Time QPCR Data Analysis Tutorial part II.flv

36 DISCUSSION Dissociation curve movie Dissociation curve results Primers used in the experiment GAPDH_Forward GAPDH_Reverse ACTIN_Forward ACTIN_Reverse Sequence (5 ->3 ) CAG CCT CAA GAT CAT CAG CA CAT GAG TCC TTC CAC GAT AC TCA CCC ACA CTG TGC CCA TCT ACG A CAG CGG AAC CGC TCA TTG CCA ATG G Product (bp) Calculated Product Tm