Real time quantitative PCR

Downloaded from genome.cshlp.org on October 20, 2016 - Published by Cold Spring Harbor Laboratory Press GENOME METHODS Real Time Quantitative PCR Christian A. Heid, 1 Junko Stevens, 2 Kenneth J. Livak, 2 and P. Mickey Williams 1'3 1BioAnalytical Technology Department, Genentech, Inc., South San Francisco, California 94080; 2Applied BioSystems Division of Perkin Elmer Corp., Foster City, California 94404 We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe {i.e., TaqMan Probe}. This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude}. Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods. Quantitative nucleic acid sequence analysis has that it be used properly for quantitation (Raey- had an important role in many fields of biologi- maekers 1995). Many early reports of quantita- cal research. Measurement of gene expression tive PCR and RT-PCR described quantitation of (RNA) has been used extensively in monitoring the PCR product but did not measure the initial biological responses to various stimuli (Tan et al. target sequence quantity. It is essential to design 1994; Huang et al. 1995a,b; Prud'homme et al. proper controls for the quantitation of the initial 1995). Quantitative gene analysis (DNA) has target sequences (Ferre 1992; Clementi et al. been used to determine the genome quantity of a 1993). particular gene, as in the case of the h u m a n HER2 Researchers have developed several methods gene, which is amplified in -30% of breast tu- of quantitative PCR and RT-PCR. One approach mors (Slamon et al. 1987). Gene and genome measures PCR product quantity in the log phase quantitation (DNA and RNA) also have been used of the reaction before the plateau (Kellogg et al. for analysis of h u m a n immunodeficiency virus 1990; Pang et al. 1990). This method requires (HIV) burden demonstrating changes in the lev- that each sample has equal input amounts of els of virus throughout the different phases of the nucleic acid and that each sample under analysis disease (Connor et al. 1993; Piatak et al. 1993b; amplifies with identical efficiency up to the point Furtado et al. 1995). of quantitative analysis. A gene sequence (con- Many methods have been described for the tained in all samples at relatively constant quan- quantitative analysis of nucleic acid sequences tities, such as [~-actin) can be used for sample (both for RNA and DNA; Southern 1975; Sharp et amplification efficiency normalization. Using al. 1980; T h o m a s 1980). Recently, PCR has conventional methods of PCR detection and proven to be a powerful tool for quantitative quantitation (gel electrophoresis or plate capture nucleic acid analysis. PCR and reverse transcrip- hybridization), it is extremely laborious to assure tase (RT)-PCR have permitted the analysis of that all samples are analyzed during the log phase minimal starting quantities of nucleic acid (as of the reaction (for both the target gene and the little as one cell equivalent). This has made pos- normalization gene). Another method, quantita- sible many experiments that could not have been tive competitive (QC)-PCR, has been developed performed with traditional methods. Although and is used widely for PCR quantitation. QC-PCR PCR has provided a powerful tool, it is imperative relies on the inclusion of an internal control competitor in each reaction (Becker-Andre 1991; Piatak et al. 1993a,b). The efficiency of each re- 3Corresponding author. action is normalized to the internal competitor. E-MAIL [email protected]; FAX (415) 225-1411. A known amount of internal competitor can be 986 ~ GENOME RESEARCH 6:986-994 9 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/96 $5.00  Downloaded from genome.cshlp.org on October 20, 2016 - Published by Cold Spring Harbor Laboratory Press REAL TIME QUANTITATIVE PCR added to each sample. To obtain relative quani- RESULTS tation, the u n k n o w n target PCR product is com- pared with the known competitor PCR product. PCR Product Detection in Real Time Success of a quantitative competitive PCR assay relies on developing an internal control that am- The goal was to develop a high-throughput, sen- plifies with the same efficiency as the target mol- sitive, and accurate gene quantitation assay for ecule. The design of the competitor and the vali- use in m o n i t o r i n g lipid-mediated therapeutic dation of amplification efficiencies require a gene delivery. A plasmid-encoding h u m a n factor dedicated effort. However, because QC-PCR does VIII gene sequence, pF8TM (see Methods), was not require that PCR products be analyzed during used as a model therapeutic gene. The assay uses the log phase of the amplification, it is the easier fluorescent Taqman methodology and an instru- of the two methods to use. m e n t capable of measuring fluorescence in real Several detection systems are used for quan- time (ABI Prism 7700 Sequence Detector). The titative PCR and RT-PCR analysis: (1) agarose Taqman reaction requires a hybridization probe gels, (2) fluorescent labeling of PCR products and labeled with two different fluorescent dyes. One detection with laser-induced fluorescence using dye is a reporter dye (FAM), the other is a quench- capillary electrophoresis (Fasco et al. 1995; Wil- ing dye (TAMRA). W h e n the probe is intact, fluo- liams et al. 1996) or acrylamide gels, and (3) plate rescent energy transfer occurs and the reporter capture and sandwich probe hybridization (Mul- dye fluorescent emission is absorbed by the der et al. 1994). Although these methods proved quenching dye (TAMRA). During the extension successful, each m e t h o d requires post-PCR ma- phase of the PCR cycle, the fluorescent hybrid- nipulations that add time to the analysis and ization probe is cleaved by the 5'-3' nucleolytic m a y lead to laboratory c o n t a m i n a t i o n . The activity of the DNA polymerase. On cleavage of sample throughput of these methods is limited the probe, the reporter dye emission is no longer (with the exception of the plate capture ap- transferred efficiently to the quenching dye, re- proach), and, therefore, these methods are not sulting in an increase of the reporter dye fluores- well suited for uses d e m a n d i n g high sample cent emission spectra. PCR primers and probes throughput (i.e., screening of large numbers of were designed for the h u m a n factor VIII se- biomolecules or analyzing samples for diagnos- quence and human/3-actin gene (as described in tics or clinical trials). Methods). O p t i m i z a t i o n reactions were per- Here we report the development of a novel formed to choose the appropriate probe and assay for quantitative DNA analysis. The assay is magnesium concentrations yielding the highest based on the use of the 5' nuclease assay first intensity of reporter fluorescent signal without described by Holland et al. (1991). The m e t h o d sacrificing specificity. The i n s t r u m e n t uses a uses the 5' nuclease activity of Taq polymerase to charge-coupled device (i.e., CCD camera) for cleave a nonextendible hybridization probe dur- measuring the fluorescent emission spectra from ing the extension phase of PCR. The approach 500 to 650 nm. Each PCR tube was monitored uses dual-labeled f l u o r o g e n i c h y b r i d i z a t i o n sequentially for 25 msec with continuous moni- probes (Lee et al. 1993; Bassler et al. 1995; Livak toring throughout the amplification. Each tube et al. 1995a,b). One fluorescent dye serves as a was re-examined every 8.5 sec. Computer soft- reporter [FAM (i.e., 6-carboxyfluorescein)] and its ware was designed to examine the fluorescent in- emission spectra is quenched by the second fluo- tensity of b o t h the reporter dye (FAM) and rescent dye, TAMRA (i.e., 6-carboxy-tetramethyl- the q u e n c h i n g dye (TAMRA). The fluorescent rhodamine). The nuclease degradation of the hy- intensity of the quenching dye, TAMRA, changes bridization probe releases the quenching of the very little over the course of the PCR amplifi- FAM fluorescent emission, resulting in an in- cation (data not shown). Therefore, the intensity crease in peak fluorescent emission at 518 nm. of TAMRA dye emission serves as an internal The use of a sequence detector (ABI Prism) allows standard with which to normalize the reporter measurement of fluorescent spectra of all 96 wells dye (FAM) emission variations. The software cal- of the thermal cycler continuously during the culates a value termed ARn (or ARQ) using the PCR amplification. Therefore, the reactions are following equation: ARn = (Rn+)-(Rn-), where monitored in real time. The output data is de- Rn § = emission intensity of reporter/emission in- scribed and quantitative analysis of input target tensity of quencher at any given time in a reac- DNA sequences is discussed below. tion tube, and R n - = emission intensitity of re- GENOME RESEARCHJ 987  Downloaded from genome.cshlp.org on October 20, 2016 - Published by Cold Spring Harbor Laboratory Press HElD ET AL. porter/emission intensity of quencher measured value remains at base line. W h e n sufficient hy- prior to PCR amplification in that same reaction bridization probe has been cleaved by the Taq tube. For the purpose of quantitation, the last polymerase nuclease activity, the intensity of re- three data points (ARns) collected during the ex- porter fluorescent emission increases. Most PCR tension step for each PCR cycle were analyzed. amplifications reach a plateau phase of reporter The nucleolytic degradation of the hybridization fluorescent emission if the reaction is carried out probe occurs during the extension phase of PCR, to high cycle numbers. The amplification plot is and, therefore, reporter fluorescent emission in- examined early in the reaction, at a point that creases during this time. The three data points represents the log phase of product accumula- were averaged for each PCR cycle and the mean tion. This is done by assigning an arbitrary value for each was plotted in an "amplification threshold that is based on the variability of the plot" shown in Figure 1A. The ARn mean value is base-line data. In Figure 1A, the threshold was set plotted on the y-axis, and time, represented by at 10 standard deviations above the mean of cycle number, is plotted on the x-axis. During the base-line emission calculated from cycles 1 to 15. early cycles of the PCR amplification, the ARn Once the threshold is chosen, the point at which Figure 1 PCR product detection in real time. (A) The Model 7700 software will construct amplification plots from the extension phase fluorescent emission data collected during the PCR amplification. The standard de- viation is determined from the data points collected from the base line of the amplification plot. Ct values are calculated by determining the point at which the fluorescence exceeds a threshold limit (usually 10 times the standard deviation of the base line). (B) Overlay of amplification plots of serially (1:2) diluted human genomic DNA samples amplified with 13-actin primers. (C) Input DNA concentration of the samples plotted versus Ct. All points represent the mean of triplicate PCR amplifications, and error bars are shown (but not always visible). 988 ~ GENOME RESEARCH  Downloaded from genome.cshlp.org on October 20, 2016 - Published by Cold Spring Harbor Laboratory Press REAL TIME QUANIIIATIVE PCR the amplification plot crosses the threshold is de- ments over a very large range of relative starting fined as CT. CT is reported as the cycle number at target quantities. this point. As will be demonstrated, the CT value is predictive of the quantity of input target. Sample Preparation Validation Several parameters influence the efficiency of CT Values Provide a Quantitative Measurement of PCR amplification: magnesium and salt concen- Input Target Sequences trations, reaction conditions (i.e., time and tem- perature), PCR target size and composition, Figure 1B shows amplification plots of 15 differ- primer sequences, and sample purity. All of the ent PCR amplifications overlaid. The amplifica- above factors are c o m m o n to a single PCR assay, tions were performed on a 1:2 serial dilution of except sample to sample purity. In an effort to h u m a n genomic DNA. The amplified target was validate the m e t h o d of sample preparation for h u m a n ~-actin. The amplification plots shift to the factor VIII assay, PCR amplification reproduc- the right (to higher threshold cycles) as the input ibility and efficiency of 10 replicate sample target quantity is reduced. This is expected be- preparations were examined. After genomic DNA cause reactions with fewer starting copies of the was prepared from the 10 replicate samples, the target molecule require greater amplification to DNA was quantitated by ultraviolet spectroscopy. degrade enough probe to attain the threshold Amplifications were performed analyzing [3-actin fluorescence. An arbitrary threshold of 10 stan- gene content in 100 and 25 ng of total genomic dard deviations above the base line was used to DNA. Each PCR amplification was performed in determine the CT values. Figure 1C represents the triplicate. Comparison of CT values for each trip- CT values plotted versus the sample dilution licate sample show minimal variation based on value. Each dilution was amplified in triplicate standard deviation and coefficient of variance PCR amplifications and plotted as mean values (Table 1). Therefore, each of the triplicate PCR with error bars representing one standard devia- amplifications was highly reproducible, demon- tion. The CT values decrease linearly with increas- strating that real time PCR using this instrumen- ing target quantity. Thus, CT values can be used tation introduces m i n i m a l variation into the as a quantitative measurement of the input target quantitative PCR analysis. Comparison of the number. It should be noted that the amplifica- mean CT values of the 10 replicate sample prepa- tion plot for the 15.6-ng sample shown in Figure rations also showed minimal variability, indicat- 1B does not reflect the same fluorescent rate of ing that each sample preparation yielded similar increase exhibited by most of the other samples. results for ~-actin gene quantity. The highest CT The 15.6-ng sample also achieves endpoint pla- difference between any of the samples was 0.85 teau at a lower fluorescent value than would be and 0.71 for the 100 and 25 ng samples, respec- expected based on the input DNA. This phenom- tively. Additionally, the amplification of each enon has been observed occasionally with other sample exhibited an equivalent rate of fluores- samples (data not shown) and may be attribut- cent emission intensity change per a m o u n t of able to late cycle inhibition; this hypothesis is DNA target analyzed as indicated by similar still under investigation. It is important to note slopes derived from the sample dilutions (Fig. 2). that the flattened slope and early plateau do not Any sample containing an excess of a PCR inhibi- impact significantly the calculated CT value as tor would exhibit a greater measured ~-actin CT demonstrated by the fit on the line shown in value for a given quantity of DNA. In addition, Figure 1C. All triplicate amplifications resulted in the inhibitor would be diluted along with the very similar CT values--the standard deviation sample in the dilution analysis (Fig. 2), altering did not exceed 0.5 for any dilution. This experi- the expected CT value change. Each sample am- m e n t contains a >100,000-fold range of input tar- plification yielded a similar result in the analysis, get molecules. Using CT values for quantitation demonstrating that this method of sample prepa- permits a much larger assay range than directly ration is highly reproducible with regard to using total fluorescent emission intensity for sample purity. quantitation. The linear range of fluorescent in- tensity measurement of the ABI Prism 7700 Se- Quantitative Analysis of a Plasmid After quence Detector only spans three logs, resulting Transient Transfection in only a 1000-fold dynamic range of input mol- ecules. Thus, CT values provide accurate measure- 293 cells were transiently transfected with a vec- GENOME RESEARCH~ 989  Downloaded from genome.cshlp.org on October 20, 2016 - Published by Cold Spring Harbor Laboratory Press HElD ET AL. Table 1. Reproducibility of Sample Preparation Method 100 ng 25 ng Sample standard standard no. Ct mean deviation CV CT mean deviation CV 1 18.24 20.48 18.23 20.55 18.33 18.27 0.06 0.32 20.5 20.51 0.03 0.1 7 2 18.33 20.61 18.35 20.59 18.44 18.37 0.06 0.32 20.41 20.54 0.11 0.54 3 18.3 20.54 18.3 20.6 18.42 18.34 0.07 0.36 20.49 20.54 0.06 0.28 4 18.15 20.48 18.23 20.44 18.32 18.23 0.08 0.46 20.38 20.43 0.05 0.26 5 18.4 20.68 18.38 20.87 18.46 18.42 0.04 0.23 20.63 20.73 0.13 0.61 6 18.54 21.09 18.67 21.04 19 18.74 0.24 1.26 21.04 21.06 0.03 0.15 7 18.28 20.67 18.36 20.73 18.52 18.39 0.12 0.66 20.65 20.68 0.04 0.2 8 18.45 20.98 18.7 20.84 18.73 18.63 0.16 0.83 20.75 20.86 0.12 0.57 9 18.18 20.46 18.34 20.54 18.36 18.29 0.1 0.55 20.48 20.51 0.07 0.32 10 18.42 20.79 18.57 20.78 18.66 18.55 0.12 0.65 20.62 20.73 0.1 0.46 Mean (1-1 O) 18.42 0.1 7 0.90 20.66 0.19 0.94 tor containing a partial cDNA for h u m a n factor between any two sample means was 0.95 CT. Ten VIII, pF8TM. A series of transfections was set nanograms of total DNA of each sample were also up using a decreasing a m o u n t of the plasmid (40, examined for [3-actin. The results again showed 4, 0.5, and 0.1 tug). Twenty-four hours post- that very similar amounts of genomic DNA were transfection, total DNA was purified from each present; the m a x i m u m mean [3-actin CT value flask of cells. [3-Actin gene quantity was chosen as difference was 1.0. As Figure 3 shows, the rate of a value for normalization of genomic DNA con- [3-actin CT change between the 100- and 10-ng centration from each sample. In this experiment, samples was similar (slope values range between [3-actin gene content should remain constant - 3 . 5 6 and -3.45). This verifies again that the relative to total genomic DNA. Figure 3 shows the m e t h o d of sample preparation yields samples of result of the [3-actin DNA measurement (100 ng identical PCR integrity (i.e., no sample contained total DNA determined by ultraviolet spectros- an excessive a m o u n t of a PCR inhibitor). How- copy) of each sample. Each sample was analyzed ever, these results indicate that each sample con- in triplicate and the mean [3-actin CT values of tained slight differences in the actual a m o u n t of the triplicates were plotted (error bars represent genomic DNA analyzed. Determination of actual one standard deviation). The highest difference genomic DNA concentration was accomplished 990 ~ GENOME RESEARCH  Downloaded from genome.cshlp.org on October 20, 2016 - Published by Cold Spring Harbor Laboratory Press REAL TIME QUANTITATIVE PCR 21.5 Sample PCR amplifications. As shown, pF8TM purified 21 from the 293 cells decreases (mean CT values in- 2 crease) with decreasing a m o u n t s of plasmid 20.5 5 transfected. The mean CT values obtained for 20- 7 pF8TM in Figure 4A were plotted on a standard .c_ 8 9 curve c o m p r i s e d of serially diluted pF8TM, 19.5- 10 oQ. shown in Figure 4B. The quantity of pF8TM, b, 19- found in each of the four transfections was de- 18.5 termined by extrapolation to the x-axis of the standard curve in Figure 4B. These uncorrected 18 1.3 1 '4 115 1'8 1'7 I'.8 1'9 .... 2.1 values, b, for pF8TM were normalized to deter- log (ng inputgenomicDNA) mine the actual a m o u n t of pF8TM found per 100 Figure 2 Sample preparation purity. The replicate ng of genomic DNA by using the equation: samples shown in Table I were also amplified in tripicate using 25 ng of each DNA sample. The fig- b x 100 ng actual pF8TM copies per ure shows the input DNA concentration (I00 and a -- 100 ng of genomic DNA 25 ng) vs. CT. In the figure, the 100 and 25 ng points for each sample are connected by a line. where a = actual genomic DNA in a sample and b = pF8TM copies from the standard curve. The normalized quantity of pF8TM per 100 ng of ge- by plotting the mean [~-actin C T value obtained nomic DNA for each of the four transfections is for each 100-ng sample on a [3-actin standard shown in Figure 4D. These results show that the curve (shown in Fig. 4C). The actual genomic quantity of factor VIII plasmid associated with DNA concentration of each sample, a, was ob- the 293 cells, 24 hr after transfection, decreases tained by extrapolation to the x-axis. with decreasing plasmid concentration used in Figure 4A shows the measured (i.e., non- the transfection. The quantity of pF8TM associ- normalized) quantities of factor VIII plasmid ated with 293 cells, after transfection with 40 pug DNA (pF8TM) from each of the four transient cell of plasmid, was 35 pg per 100 ng genomic DNA. transfections. Each reaction contained 100 ng of This results in -520 plasmid copies per cell. total sample DNA (as determined by UV spectros- copy). Each sample was analyzed in triplicate DISCUSSION 26 w e have described a new m e t h o d for quantitat- -- y = 27.73 + -3.52x R= 1 ing gene copy numbers using real-time analysis -- y = 28.77 + -3.56x R= 1 25- of PCR amplifications. Real-time PCR is compat- ible with either of the two PCR (RT-PCR) ap- ~- 24- O proaches: (1) quantitative competitive where an .__ "~ 23- internal competitor for each target sequence is used for normalization (data not shown) or (2) 22- quantitative comparative PCR using a normaliza- i FS~ 'ri!:fecteransfected~;i 21 = 4.0 Ilg | tion gene contained within the sample (i.e., 6-ac- tin) or a "housekeeping" gene for RT-PCR. If 20 equal amounts of nucleic acid are analyzed for 0.8 ~ 1'.2 114 116 118 2.2 each sample and if the amplification efficiency log (ng input DNA) before quantitative analysis is identical for each Figure 3 Analysisof transfected cell DNA quantity sample, the internal control (normalization gene and purity. The DNA preparations of the four 293 or competitor) should give equal signals for all cell transfections (40, 4, 0.5, and 0.1 ~g of pF8TM) samples. were analyzed for the 13-actin gene. 100 and 10 ng The real-time PCR method offers several ad- (determined by ultraviolet spectroscopy) of each sample were amplified in triplicate. For each vantages over the other two methods currently amount of pF8TM that was transfected, the 13-actin employed (see the introduction). First, the real- CT values are plotted versus the total input DNA time PCR method is performed in a closed-tube concentration. system and requires no post-PCR manipulation GENOME RESEARCHJ 991  Downloaded from genome.cshlp.org on October 20, 2016 - Published by Cold Spring Harbor Laboratory Press HElD ET AL. 28 30, o. ~-~40..155+ -3.71x 26- 25-1 24 22- I-- 20J ~ 2o 18- 15J 16- 14 lO-~ 4'o 4'.o o'.s o'.1 Plasmid used in transfection (lig) log (relative factor VIII copies) < D 32 .o i n7 30 Eo 28 26 o 24- o 22 20- 18 ._~ -2 -1 40 ug 4 ug 0.5 ug 0.1 ug log (ng input genomic DNA) n- plasmid used in transfection 01g) Figure 4 Quantitative analysis of pF8TM in transfected cells. (A) Amount of plasmid DNA used for the transfection plotted against the mean Ct value deter- mined for pF8TM remaining 24 hr after transfection. (B,C) Standard curves of pF8TM and 13-actin, respectively, pF8TM DNA (B) and genomic DNA (C) were diluted serially 1:5 before amplification with the appropriate primers. The 13-actin standard curve was used to normalize the results of A to 100 ng of genomic DNA. (D) The amount of pF8TM present per 100 ng of genomic DNA. of sample. Therefore, the potential for PCR con- for each sample minimizing potential error. The tamination in the laboratory is reduced because system allows for a very large assay dynamic amplified products can be analyzed and disposed range (approaching 1,000,000-fold starting tar- of without opening the reaction tubes. Second, get). Using a standard curve for the target of in- this method supports the use of a normalization terest, relative copy number values can be deter- gene (i.e., [3-actin) for quantitative PCR or house- mined for any u n k n o w n sample. Fluorescent keeping genes for quantitative RT-PCR controls. threshold values, CT, correlate linearly with rela- Analysis is performed in real time during the log tive DNA copy numbers. Real time quantitative phase of product accumulation. Analysis during RT-PCR methodology (Gibson et al., this issue) log phase permits many different genes (over a has also been developed. Finally, real time quan- wide input target range) to be analyzed simulta- titative PCR methodology can be used to develop neously, without concern of reaching reaction high-throughput screening assays for a variety of plateau at different cycles. This will make multi- applications [quantitative gene expression (RT- gene analysis assays much easier to develop, be- PCR), gene copy assays (Her2, HIV, etc.), geno- cause individual internal competitors will not be typing (knockout mouse analysis), and immuno- needed for each gene u n d e r analysis. Third, PCR]. sample t h r o u g h p u t will increase dramatically Real-time PCR may also be performed using with the new method because there is no post- intercalating dyes (Higuchi et al. 1992) such as PCR processing time. Additionally, working in a e t h i d i u m b r o m i d e . The f l u o r o g e n i c probe 96-well format is highly compatible with auto- m e t h o d offers a major advantage over inter- mation technology. calating dyes--greater specificity (i.e., primer The real-time PCR method is highly repro- dimers and nonspecific PCR products are not de- ducible. Replicate amplifications can be analyzed tected). 992 ~ GENOME RESEARCH  Downloaded from genome.cshlp.org on October 20, 2016 - Published by Cold Spring Harbor Laboratory Press REAL TIME QUANTITATIVE PCR METHODS reactions were performed in the Model 7700 Sequence De- tector (PE Applied Biosystems), which contains a Gene- Generation of a Plasmid Containing a Partial Amp PCR System 9600. Reaction conditions were pro- cDNA for Human Factor VIII grammed on a Power Macintosh 7100 (Apple Computer, Santa Clara, CA) linked directly to the Model 7700 Se- Total RNA was harvested (RNAzol B from Tel-Test, Inc., quence Detector. Analysis of data was also performed on Friendswood, TX) from cells transfected with a factor VIII the Macintosh computer. Collection and analysis software expression vector, pCIS2.8c25D (Eaton et al. 1986; Gor- was developed at PE Applied Biosystems. man et al. 1990). A factor VIII partial cDNA sequence was generated by RT-PCR [GeneAmp EZ tTth RNA PCR Kit (part N808-0179, PE Applied Biosystems, Foster City, CA)] Transfection of Cells with Factor Vlll Construct using the PCR primers F8for and F8rev (primer sequences are shown below). The amplicon was reamplified using Four T175 flasks of 293 cells (ATCC CRL 1573), a human modified F8for and F8rev primers (appended with BamHI fetal kidney suspension cell line, were grown to 80% con- and HindIII restriction site sequences at the 5' end) and fluency and transfected pF8TM. Cells were grown in the cloned into pGEM-3Z (Promega Corp., Madison, WI). The following media: 50% HAM'S F12 without GHT, 50% low resulting clone, pF8TM, was used for transient transfection glucose Dulbecco's modified Eagle medium (DMEM) with- of 293 cells. out glycine with sodium bicarbonate, 10% fetal bovine serum, 2 mM L-glutamine, and 1% penicillin-streptomy- cin. The media was changed 30 min before the transfec- Amplification of Target DNA and Detection of tion. pF8TM DNA amounts of 40, 4, 0.5, and 0.1 txg were Amplicon Factor VIII Plasmid DNA added to 1.5 ml of a solution containing 0.125 M CaC12 and 1 • HEPES. The four mixtures were left at room tem- (pF8TM) was amplified with the primers F8for 5'-CCC- perature for 10 min and then added dropwise to the cells. GTGCCAAGAGTGACGTGTC-3' and F8rev 5'-AAACCT- The flasks were incubated at 37~ and 5% CO 2 for 24 hr, CAGCCTGGATGGTAGG-3'. The reaction produced a 422- washed with PBS, and resuspended in PBS. The resus- bp PCR product. The forward primer was designed to rec- pended cells were divided into aliquots and DNA was ex- ognize a unique sequence found in the 5' untranslated tracted immediately using the QIAamp Blood Kit (Qiagen, region of the parent pCIS2.8c25D plasmid and therefore Chatsworth, CA). DNA was eluted into 200 ixl of 20 mM does not recognize and amplify the h u m a n factor VIII Tris-HC1 at pH 8.0. gene. Primers were chosen with the assistance of the com- puter program Oligo 4.0 (National Biosciences, Inc., Ply- mouth, MN). The human [3-actin gene was amplified with ACKNOWLEDGMENTS the primers ~-actin forward primer 5'-TCACCCACACTGT- GCCCATCTACGA-3' and [3-actin reverse primer 5'-CAG- We thank Genentech's DNA Synthesis Group for primer CGGAACCGCTCATTGCCAATGG-3'. The reaction pro- synthesis and Genentech's Graphics Group for assistance duced a 295-bp PCR product. with the figures. Amplification reactions (50 /xl) contained a DNA The publication costs of this article were defrayed in sample, 10• PCR Buffer II (5 ~l), 200 bi.M dATP, dCTP, part by payment of page charges. This article must there- dGTP, and 400 txM dUTP, 4 mM MgC12, 1.25 Units Ampli- fore be hereby marked "advertisement" in accordance Taq DNA polymerase, 0.5 unit AmpErase uracil N-gly- with 18 USC section 1734 solely to indicate this fact. cosylase (UNG), 50 pmole of each factor VIII primer, and 15 pmole of each ~-actin primer. The reactions also contained one of the following detection probes (100 nM each): REFERENCES F8probe 5 '(FAM)AGCTCTCCACCTGCTTCTTTCTGT- Bassler, H.A., S.J. Flood, K.J. Livak, J. Marmaro, R. Knorr, GCCTT(TAMRA)p-3' and [3-actin probe 5'-(FAM)ATGCCC- and C.A. Batt. 1995. Use of a fluorogenic probe in a X(TAMRA)CCCCCATGCCATCp-3' where p indicates PCR-based assay for the detection of Listeria phosphorylation and X indicates a linker arm nucleotide. monocytogenes. App. Environ. 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Towards 1996. 994 ~ GENOME RESEARCH  Downloaded from genome.cshlp.org on October 20, 2016 - Published by Cold Spring Harbor Laboratory Press Real time quantitative PCR. C A Heid, J Stevens, K J Livak, et al. Genome Res. 1996 6: 986-994 Access the most recent version at doi:10.1101/gr.6.10.986 References This article cites 28 articles, 12 of which can be accessed free at: http://genome.cshlp.org/content/6/10/986.full.html#ref-list-1 Creative This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the Commons first six months after the full-issue publication date (see License http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at http://creativecommons.org/licenses/by-nc/3.0/. Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the Service top right corner of the article or click here. 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