PCR結(jié)果分析CT法詳細(xì)介紹_圖文

1、 or1.THE 2C T METHODX N (1E C T K ,6Methodwhere X N is equal to the normalized amount of target The equation that describes the exponential amplifi-(X 0/R 0and C T is equal to the difference in threshold cation of PCR iscycles for target and reference (C T ,X C T ,R .Rearranging gives the expression

2、X n X 0(1E X n ,1X N K (1E C T .7where X n is the number of target molecules at cycle The final step is to divide the X N for any sample q by n of the reaction,X 0is the initial number of target the X N for the calibrator (cb:molecules.E X is the efficiency of target amplification,and n is the numbe

3、r of cycles.The threshold cycle (C T indicates the fractional cycle number at which the X N,q X N,cb K (1E C T,qK (1E C T,cb (1E C T.8amount of amplified target reaches a fixed threshold.Thus,Here C T (C T ,q C T ,cb .For amplicons designed to be less than 150bp and for X T X 0(1E X C T,X K X2which

4、the primer and Mg 2+concentrations have been properly optimized,the efficiency is close to one.There-where X T is the threshold number of target molecules,fore,the amount of target,normalized to an endogenous C T ,X is the threshold cycle for target amplification,and reference and relative to a cali

5、brator,is given byK X is a constant.A similar equation for the endogenous reference (internal control genereaction isamount of target 2C T .9R T R 0(1E R C T,R K R ,3is a constant.Dividing X T by R T gives the expressionX T R T X 0(1E X C T,X R 0(1E R C T,R K XK RK .4cation of the target gene (c-myc

6、 and internal control (GAPDHwas examined using real-time PCR and TaqMan detection.Using reverse transcriptase,cDNA was synthesized from 1g total RNA isolated E X E R E ,from human Raji cells.Serial dilutions of cDNA were amplified by real-time PCR using gene-specific primers.The most concentrated sa

7、mple contained cDNA derived from 1ng of total RNA.The C T (C T ,c myc C T ,GAPDH was calculated for each cDNA dilution.The data X 0R 0(1E C T,X C T,R K ,5were fit using least-squares linear regression analysis (N 3.看擴(kuò)增效率是否相同丆就是看代爾塔CT 是否隨著模板稀釋程度改變而改變算出代爾塔CT,作圖丆斜率越接近于1C 說(shuō)明目的基因和內(nèi)參擴(kuò)增效率一致丆可以用這種方法time zer

8、o,was calculated for each sample using Eq.9The choice of calibrator for the 2C Tmethod de-pends on the type of gene expression experiment that (Fig.2,column 9.The mean,SD,and CV are then determined from the triplicate samples at each time one has planned.The simplest design is to use the un-treated

9、control as the calibrator.Using the 2C T point.Using this analysis,the value of the mean fold change at time zero should be very close to one (i.e.,method,the data are presented as the fold change in gene expression normalized to an endogenous reference since 201.We have found the verification of th

10、e mean fold change at time zero to be a convenient method gene and relative to the untreated control.For the un-treated control sample,C T equals zero and 20equals to check for errors and variation among the triplicate samples.A value that is very different from one sug-one,so that the fold change i

11、n gene expression relative to the untreated control equals one,by definition.For gests a calculation error in the spreadsheet or a very high degree of experimental variation.the treated samples,evaluation of 2C T indicates the fold change in gene expression relative to the untreated In the preceding

12、 example,three separate RNA prepa-rations were made for each time point and carried control.Similar analysis could be applied to study the time course of gene expression where the calibrator through the analysis.Therefore,it made sense to treat each sample separately and average the results aftersam

13、ple represents the amount of transcript that is ex-pressed at time zero.the 2C T calculation.When replicate PCRs are run on the same sample,it is more appropriate to averageSituations exist where one may not compare the 如果目的基因和內(nèi)參擴(kuò)增效率不一致丆那么需要用標(biāo)準(zhǔn)品做絕對(duì)定量。或者重新設(shè)計(jì)引物使擴(kuò)增效率一致最終的結(jié)果是對(duì)照組為1C 實(shí)驗(yàn)組是對(duì)照組的幾倍或者幾分之幾不同種類(lèi)細(xì)

14、胞丆目的基因和內(nèi)參的表達(dá)都會(huì)不同用對(duì)照組的值發(fā)現(xiàn)錯(cuò)誤或者三組數(shù)據(jù) 變異程度cDNA 和CT 做圖丆如果斜率接近于0C 說(shuō)明目的基因和內(nèi)參的擴(kuò)增效率是一致的丆可以用這種方法C T data before performing the 2C T calculation.Ex-(GAPDHwere amplified in separate wells.There is no reason to pair any particular c-myc well with any actly how the averaging is performed depends on if thetarget an

15、d reference are amplified in separate wells particular GAPDH well.Therefore,it makes sense to average the c-myc and GAPDH C T values separately or in the same well.Table 1presents data from an experiment where the target (c-myc and referencebefore performing the C T calculation.The varianceTABLE 1Tr

16、eatment of Replicate Data Where Target and Reference Are Amplified in Separate Wells aC T (Avg.c-myc C T C T (Avg.C T Normalized c-myc amount Tissue c-myc C T GAPDH C TAvg.GAPDH C TAvg.C T ,Brain relative to brain 2C TBrain1.0(0.91.1Kidney5.6(5.36.0aTotal RNA from human brain and kidney were purchas

17、ed from Clontech.Using reverse transcriptase,cDNA was synthesized from 1g total RNA.Aliquots of cDNA were used as template for real-time PCR reactions containing either primers and probe for c-myc or primers and probe for GAPDH.Each reaction contained cDNA derived from 10ng total RNA.Six replicates

18、of each reaction were performed.均值+標(biāo)準(zhǔn)差可通過(guò)SPSS 求的每一組數(shù)都相減丆所以又得到一組數(shù)值丆再求均值+標(biāo)準(zhǔn)差求平均值我算出來(lái)的值是2.144C 可能和前面小數(shù)點(diǎn)的取舍=0.228/1.02是否需要計(jì)算平均值取決于目的基因和內(nèi)參是否在同一管或者不同管內(nèi)擴(kuò)增。ticular well,we know that the c-myc reaction and the GAPDH reaction had exactly the same cDNA input.Normalizing to an endogenous reference provides a me

19、thod for correcting results for differing amounts of Therefore,it makes sense to calculate C T separately for each well.These C T values can then be averaged input RNA.One hallmark of the 2C T method is that it uses data generated as part of the real-time PCR before proceeding with the 2C T calculat

20、ion.Again,the estimated error is given as an asymmetric range of experiment to perform this normalization function.This is particularly attractive when it is not practical values,reflecting conversion of an exponential variable to a linear comparison.to measure the amount of input RNA by other metho

21、ds.Such situations include when only limited amounts of In Tables 1and 2,the estimated error has not been increased in proceeding from the C T column to the RNA are available or when high-throughput processing of many samples is desired.It is possible,though,to C T column.This is because we have dec

22、ided to dis-play the data with error shown both in the calibrator normalize to some measurement external to the PCR experiment.The most common method for normaliza-and in the test sample.Subtraction of the average C T ,cb to determine the C T value is treated as subtraction tion is to use UV absorba

23、nce to determine the amountTABLE 2Treatment of Replicate Data Where Target and Reference are Amplified in the Same Well ac-myc C T (Avg.c-myc C T C T (Avg.C T Normalized c-myc amount Tissue C T GAPDH C TAvg.GAPDH C T Avg.C T ,Brain relative to brain 2C TBrainKidney24.184.52AverageaAn experiment like

24、 that described in Table 1was performed except the reactions contained primers and probes for both c-myc and GAPDH.The probe for c-myc was labeled with the reporter dye FAM and the probe for GAPDH was labeled with the reporter dye JOE.Because of the different reporter dyes,the real-time PCR signals

25、for c-myc and GAPDH can be distinguished even though both amplifications are occurring in the same well.在相同的管子里擴(kuò)增丆需要在探針上標(biāo)上不同顏色的熒光物質(zhì)用不同探針進(jìn)行標(biāo)記的丆可以使內(nèi)參和目的基因在同意管內(nèi)擴(kuò)增。ANALYSIS OF REAL-TIME PCR DATA 407 of RNA added to a cDNA reaction. PCRs are then set up using cDNA derived from the same amount of input RN

26、A. One example of using this external normalization is to study the effect of experimental treatment on the expression of an endogenous reference to determine if the internal control is affected by treatment. Thus, the target gene and the endogenous reference are one in the same. In this case, Eq. 2

27、 is not divided by Eq. 3 and Eq. 5 becomes X0 (1 EXCT,X KX. Rearranging gives the expression X0 KX (1 EXCT,X. 11 10 equation where C T CT,Time x CT,Time 0 (Fig. 3. A statistically significant relationship exists between the treatment and expression of GAPDH but not for 2microglobulin (Fig. 3. Theref

28、ore, 2-microglobulin makes a suitable internal control in quantitative serum stimulation studies while GAPDH does not. This exam method may be used ple demonstrates how the 2CT to analyze relative gene expression data when only one gene is being studied. 3. STATISTICAL ANALYSIS OF REAL-TIME PCR DATA

29、 The endpoint of real-time PCR analysis is the threshold cycle or CT. The CT is determined from a loglinear plot of the PCR signal versus the cycle number. Thus, CT is an exponential and not a linear term. For this reason, any statistical presentation using the raw CT Now, dividing X0 for any sample

30、 q by the X0 for the calibrator (cb gives X0,q KX (1 EXCT,q , (1 EXCT X0,cb KX (1 EXCT,cb 12 where C T is equal to CT,q CT,cb. The prime is used to distinguish this expression from the previous CT calculation (see Eq. 6 that involved subtraction of CT values for target and reference. As stated in Se

31、ction 1.1, if properly optimized, the efficiency is close to one. The amount of endogenous reference relative to a calibrator then becomes . 2CT 13 Method 2.2. Application of the 2CT method is to An appropriate application of the 2CT determine the effect of the experimental treatment on the expressi

32、on of a candidate internal control gene. To demonstrate this analysis, serum starvation and induction experiments were performed (7. Serum starvation/ induction is a commonly used model to study the decay of certain mRNAs (8. However, serum may alter the expression of numerous genes including standa

33、rd housekeeping genes (9. Gene expression was induced in NIH 3T3 cells by adding 15% serum following a 24-h period of serum starvation. Poly(A+ RNA was extracted from the cells and equivalent amounts were converted to cDNA. The amounts of 2-microglobulin and GAPDH cDNA were determined by real-time q

34、uantitative PCR with SYBR Green detection (7. The relative amounts of 2-micro globulin and GAPDH are presented using the 2CT FIG. 3. Application of the 2CT method. The following experiment was conducted to validate the effect of treatment on the expression of candidate internal control genes. NIH 3T

35、3 fibroblasts were serum starved for 24 h and then induced with 15% serum over an 8-h period. Samples were collected at various times following serum stimulation; mRNA was extracted and converted to cDNA. The cDNA was subjected to real-time quantitative PCR using gene-specific primers for 2-microglo

36、bulin and GAPDH. The fold change in gene expression was calculated using Eq. 13, where CT (CT,Time x CT,Time 0 and is presented for both 2-microglobulin (A and GAPDH (B. Reprinted from T. D. Schmittgen and B. A. Zakrajsek (2000 Effect of experimental treatment on housekeeping gene expression: Valida

37、tion by realtime quantitative RT-PCR, J. Biochem. Biophys. Methods 46, 6981, with permission of Elsevier Science. 408 LIVAK AND SCHMITTGEN 本頁(yè)已使用福昕閱讀器進(jìn)行編輯。 福昕軟件（），版權(quán)所有， 僅供試用。 values should be avoided. As described within the previous sections of this article, presentation of relative PCR data is most

38、 often calculated along with an internal control and/or calibrator sample and is rarely presented as the CT . An exception is when one is interested in examining the sample-to-sample variation among replicate reactions. 單獨(dú)說(shuō)CT是不準(zhǔn)確的丆需要和內(nèi)參進(jìn)行對(duì)比 To demonstrate this, 96 replicate reactions of the identica

39、l cDNA were performed using real-time PCR and SYBR Green detection. A master mixture containing all of the ingredients was pipetted into individual tubes of a 96-well reaction plate. The samples were subjected to real-time PCR and the individual CT values were determined. To examine the intrasample

40、variation, the mean SD was determined from the 96 samples. If calculated from the raw CT the mean SD was 20.0 0.194 with a CV of 0.971%. However, when the individual CT values were converted to the linear form using the term 2C T, the mean SD was 9.08 107 1.33 107 with a CV of 13.5%. As demonstrated

41、 by this simple example, reporting the data obtained from the raw CT values falsely represents the variation and should be avoided. Converting the individual data to a linear form using 2C T more accurately depicts the individual variation among replicate reactions. should be used. Otherwise, presen

42、tation of the relative gene expression should suffice. Relative quantification may be easier to perform than the absolute method because the use of standard curves is not required. The equations provided herein should be sufficient for an investigator to analyze quantitative gene expression data usi

43、ng relative quantification. To summarize the important steps in the design and evaluation of the experiment: (i select an internal control gene, (ii validate the internal control to determine that it is not affected by experimental treatment, and (iii PCR on perform dilutions of RNA or cDNA for both the target and internal control genes to ensure that the efficiencies are similar. Finally, statistical data should be converted to the linear form by the 2C T calculation and should not be presented by the raw CT values. REFERENCES 1. M