Board of Trustees of the Leland Stanford Junior University v. Roche Molecular Systems, Inc. et al

Filing 187

Board of Trustees of the Leland Stanford Junior University v. Roche Molecular Systems, Inc. et al

Doc. 187 Att. 11

Case 3:05-cv-04158-MHP

Document 187-12

Filed 08/29/2007

Page 1 of 9

EXHIBIT K

Dockets.Justia.com

Downloaded from www.genome.org on August 26, 2007

Case 3:05-cv-04158-MHP

Document 187-12

Filed 08/29/2007

Page 2 of 9

A novel method for real time quantitative RT-PCR.
U E Gibson, C A Heid and P M Williams Genome Res. 1996 6: 995-1001 Access the most recent version at doi:10.1101/gr.6.10.995

References

This article cites 13 articles, 4 of which can be accessed free at: http://www.genome.org#References Article cited in: http://www.genome.org/cgi/content/abstract/6/10/995#otherarticles

Email alerting service

Receive free email alerts when new articles cite this article - sign up in the box at the top right corner of the article or click here

Notes

To subscribe to Genome Research go to: http://www.genome.org/subscriptions/

© 1996 Cold Spring Harbor Laboratory Press

Downloaded from www.genome.org on August 26, 2007

Case 3:05-cv-04158-MHP

Document 187-12
GENOME METHODS

Filed 08/29/2007

Page 3 of 9

A Novel Method for Real Time Quantitative RT-PCR
Ursula E.M. Gibson, 1 Christian A. Heid, and P. Mickey Williams
Genentech, Inc., South San Francisco, California 94080-4990 A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR} using real time detection and the S' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR} target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.

The polymerase chain reaction (PCR) is a rapid and powerful technique for the in vitro amplification of DNA (Mullis et al. 1986; Gibbs 1990). Quantitative PCR (QC PCR) has been used to quantitate small amounts of DNA and QC RTPCR has been used to measure mRNA. QC PCR and QC RT-PCR methods use an internal control that is coamplified with the target sequence (Becker-Andre 1991; Ferre 1992; Siebert and Larrick 1992; Piatak et al. 1993; McCulloch et al. 1995; Raeymaekers 1995). The internal control can be designed several ways: scrambling of the internal sequence, mutation of the target amplicon, deletion or insertion of sequences into the target amplicon, or splicing of the target primer sequences onto a n o n h o m o l o g o u s DNA sequence. As a general rule, the target and control should use the same primers, contain similar guanine + cytosine (G + C) content, and be of equal or similar length. Once a control has been designed, it is important to validate the internal control. Validation requires demonstration that the competitive control amplify with equal efficiency and achieve plateau simultaneously with the target (Raeymaekers 1995). Although absolute quantitation requires the accurate determination of internal control concentration, relative quantitation can be established easily with a validated internal control. Quantitation of target and
1Corresponding author. E-MAIL [e-mail]; FAX [phone].

coamplified internal control generally is done by quantitating the intensity of ethidium bromide staining of the PCR products on an agarose or acrylamide gel, a time-consuming and potentially inaccurate approach. The 5' nuclease assay for detecting PCR products (Holland et al. 1991; Lee et al. 1993; Livak et al. 1995a,b) uses a nonextendable oligonucleotide hybridization probe. The probe is labeled with a reporter fluorescent dye [FAM (6-carboxyfluorescein)] at the 5' end and a quencherfluorescent dye [TAMRA (6-carboxy-tetramethylrhodamine)] at the 3' end. When the probe is intact, the reporter dye emission is quenched owing to the physical proximity of the reporter and quencher fluorescent dyes. During the extension phase of the PCR cycle, however, the nucleolytic activity of the DNA polymerase cleaves the hybridization probe and releases the reporter dye from the probe. The resulting relative increase in reporter fluorescent dye emission is monitored in real time during PCR amplification using a sequence detector, the 7700 Sequence Detector (PE Applied BioSystems, Foster City, CA). The sequence detector is a combination thermal cycler, laser, and detection and software system that automates 5' nuclease-based detection and quantiration of nucleic acid sequences. Fluorescence intensity produced during PCR amplifications in each of the 96 tubes is monitored in real time. A computer algorithm compares the amount of reporter dye emission (R) with the quenching dye

6:995-1001 9

by Cold Spring Harbor Laboratory Press ISSN 1054-9803/96 $5.00

GENOME RESEARCH@995

Downloaded from www.genome.org on August 26, 2007

Case 3:05-cv-04158-MHP
GIBSON ET AL.

Document 187-12

Filed 08/29/2007

Page 4 of 9

emission (Q) every 8.5 seconds during the PCR amplification, generating a ARn value (R/Q) (also called ARQ). The ARn value reflects the a m o u n t of hybridization probe that has been degraded. The algorithm fits an exponential function to the mean ARn values of the last three data points of every PCR extension cycle, generating an amplification plot. A relative fluorescent emission threshold is set based on the baseline of the ARn during the first 10-15 cycles (Held et al., this issue). The algorithm calculates the cycle at which each PCR amplification reaches a significant (i.e. usually 10 times the standard deviation of the baseline) threshold (CT). In the accompanying manuscript (Heid et al., this issue), it was demonstrated that the calculated CT value is proportional to the n u m b e r of target copies present in the sample. Thus, the CT value is a quantitative measurement of the copies of the target found in any sample. The use of a charge coupled device (CCD) camera permits the detection of a wide spectrum of emission wavelengths. By using target and control probes containing different reporter fluorescent dyes [FAM, JOE (2,7-dimethoxy-4,Sdichloro-6-carboxy-fluorescein), or TET (tetrachloro-6-carboxy-fluorescein)], it is possible to detect simultaneously both target and control RNA in a single reaction tube. However, in practice this approach is limited to concentrations of target and internal control RNA or DNA that are within 1000-fold of each other. This limitation is attributable, at least partially, to the overlapping spectra of the reporter dyes available. To develop an assay with a large dynamic range of input target quantitation, duplicate reactions containing both target and internal control RNA were set up. Target probe (prA) was added to one reaction and internal control probe (prB) to the other reaction. With this approach accurate detection of low concentrations of target mRNA without interference from internal control fluorescence was possible. This report describes a quantitative RT-PCR assay, that was developed to support a gene therapy project aimed at treating cystic fibrosis. The target for this assay was the cystic fibrosis transmembrane receptor (CFTR) mRNA, and the internal control was constructed by adding the CFTR forward and reverse primers to a sequence of the pGEM-3Z plasmid. The pGEM-3Z plasmid sequence was of similar length and contained similar G + C nucleotide content to the CFTR amplification product. 996 @GENOME RESEARCH

RESULTS
Amplification Plots and Cycle Threshold, CT During the PCR amplification, the nucleolytic activity of the DNA polymerase (Tfl) cleaves the target specific hybridization probe and releases the reporter dye, FAM or TET, from the probe. The increase in fluorescence emission of the reporter dye is proportional to the a m o u n t of PCR product accumulated, which in turn is proportional to the starting target concentration (Heid et al., this issue). Fluorescence emission is monitored, in real time using a sequence detector. The emission intensity of the quencher dye, TAMRA, which remains relatively constant during amplification (Livak et al. 1995a), was used as an internal control to normalize fluorescence emission and calculate the kRn (reporter dye emission/ quencher dye emission). ARn is the fluorescence signal increase due to template amplification and is calculated by subtracting the background fluorescence: ARn = (Rn+) - (Rn-), where Rn+= (emission intensity of reporter)/(emission intensity of q u e n c h e r of PCR w i t h t e m p l a t e ) a n d Rn- = (emission intensity of reporter)/(emission intensity of quencher of PCR without template). Figure 1 shows amplification plots of twofold serial dilutions of total RNA from adeno CFTRinfected T84 cells. For this experiment total RNA was serially diluted from a starting concentration that contained -2 x 1 0 7 copies of adeno CFTR mRNA d o w n to a c o n c e n t r a t i o n c o n t a i n i n g -1000 copies (the mRNA copy values were deter-

Copies
7-

65g 4-

o~X + § A -@A AA @ 0 ~ · +~ ~ A e e e e e e e A O0 + 9 ~ : i i i i II II II IIII , ox II e , 0 [3 + AAAA 9 ~, 9 1 4 9 1 4 9 1 4 9 1 4"9 O~ 1 7 6 2 1 5 AT 9
oo~
ox

o8~ ~

++_+$$~$

·

2.s~6

1.25e6 6.25e5 3.12e5 1.56e5 7.81e4 3.91e4 1.95e4
9.77e3

" 9149

oelI_A o E j ~ r ~ E ~ ~ E)[][]OD[]E][][]I3[]

9

o

0000000

0
[]

i~ []
[ I

4.88e3 2.44e3 1.22e3

2

I

I

I

I

I

I

I

I

I

I

I

I

I

14

18

22

26 30 Cycles

34

38

F i g u r e 1 Amplification plots of adeno-CFTR target mRNA. Twofold serial dilutions of adeno-CFTR target mRNA were reverse transcribed and amplified using the 5' nuclease assay and the sequence detector. For each dilution the ARn is plotted against the cycle number.

Downloaded from www.genome.org on August 26, 2007

Case 3:05-cv-04158-MHP

Document 187-12

Filed 08/29/2007

Page 5 of 9

A NOVEL METHOD FOR REAL TIME QUANTITAIIVE RT-PCR mined comparing the T84 cell total RNA with a known a m o u n t of internal control RNA copies). The dilutions were then reverse transcribed and amplified using RT-PCR. The change in ARn is proportional to the change in concentration of the amplified target, so that dilutions of target require additional PCR cycles to raise the ARn above the threshold value, CT. of target and internal control are the same over the range tested. Real Time QC RT-PCR A k n o w n a m o u n t of an internal control was added for reverse transcription and amplification. By amplifying both internal control and target in the same tube, identical conditions for each are assured. For this purpose, a mixture that contained all the reagents required for reverse transcription and amplification (RT-PCR), primers P1 and P2, and target samples (containing unknown amounts of target CFTR mRNA), was prepared. This mixture was used to generate two sets of tubes, set I and set II, containing eight serial dilutions of a known a m o u n t of internal control RNA. To detect the a m o u n t of the CFTR mRNA RT-PCR amplicon, the CFTR target hybridization probe was added to set I tubes and the internal control hybridization probe was added to set II tubes. Both sets of tubes were subjected to RTPCR amplification using the sequence detector. Because the corresponding tubes from sets I and II contained identical concentrations of target and internal control RNA, reverse transcription and amplification for both sets of tubes were assumed to be identical. However, because the set I tubes contained the target probe, its reporter fluorescent emission was attributable entirely to amplification of target and unaffected by the concentration of internal control RNA. The reporter fluorescence in the tubes containing internal control probe (set II) was attributable entirely to internal control amplification. By plotting the m e a n CT values of internal control and target against the known internal control copy number, the n u m b e r of u n k n o w n target molecules could be determined from the theoretical equivalence point, where CT of target equals CT of internal control (i.e. where the lines intersect; see Figs. 3A, B). In this experiment, two different concentrations of target, either a 1:25 dilution (Fig. 3A) or a 1:125 dilution (Fig. 3B) of the total RNA preparation from adenovirus-infected cells, were used with twofold serial dilutions of internal control. As shown in Figure 3A, the mean C T of the internal control dilution series intersected the m e a n CT plot of the 1:25 dilution of target at an estimated 6.7 · 1 0 6 copies of target mRNA/ml, whereas the 1:125 dilution of target indicated 9.6 · 10 s copies of target mRNA/ml (Fig. 3B). Multiplication by their respective dilution factors gave 1.7 · 108 copies of mRNA/ml for the 1:25

Colinearity of Dilution of Target CFTR mRNA and Internal Control RNA Colinearity of dilution was determined by making serial twofold dilutions of both adeno CFTR internal control and total cell RNA containing the adeno CFTR target mRNA. The range of internal control dilutions covers 6.5 logs and the range of target covers 4 logs of input molecules. Both target and internal control dilutions were reverse transcribed, amplified as described in Methods, and the CT values were calculated and plotted against the relative a m o u n t of internal control RNA. Each point of the curve (Fig. 2) represents the mean of the three separate RT-PCR amplifications depicted with error bars (too small to be visible in the graph) representing one standard deviation and shows the CT value increasing by approximately one for each twofold dilution. Figure 2 shows that b o t h target adeno CFTR mRNA and internal control adeno CFTR RNA amplify linearly and that amplification efficiencies

40~. 35 ~ y = 47.83 + -3.306log(x) R= 0.9993 - y = 46.22 + -3.125log(x) R= 0.9984

o~ 30
25-

~'~[]

2015103 ~ C TTarget ]104
i 06 T i i i

10 s

1

10 ~

10 e

109

101~

RelativeCopies

Figure 2 Colinearity of dilution and assay range of adeno CFTR target and internal control RNA. Twofold serial dilutions of target and internal control RNA were prepared in triplicate, reverse transcribed, and amplified using the sequence detector. Mean Ct values are plotted against relative copy number, with error bars representing one standard deviation.

GENOME RESEARCHJ 997

Downloaded from www.genome.org on August 26, 2007

Case 3:05-cv-04158-MHP
GIBSON ET AL.

Document 187-12

Filed 08/29/2007

Page 6 of 9

A
22

A
21 20 19 18
o

C

~
' ' ' ' ''"1 ' ' ''

= w-

Mean

Ct

Competitor

Mean C t Target

17 16

- y = 20.4 + -0.3591log(x) R= 0.951
''"1 , , r,,,, j

14 10 5

106

10 z

10 s

Relative CompetitorCopies
2423222120-~ 19
. . . . .Ox Oe,

of each dilution of adeno CFTR target mRNA and internal control RNA were reverse transcribed and amplified on three separate days as described. The mean CT values, standard deviation (S.D.) and the coefficient of variation (CV) were calculated for each day to obtain intra-assay precision. Mean CT values of target were found to range from 29.3 to 29.6 with an intra-assay precision of target amplification from 0.4% to 1.3% CV (S.D. 0.092 to 0.372). Mean internal control CT values ranged from 17.4 to 18.3 and precision of amplification ranged from 0.6 to 1.7% (S.D. 0.116 to 0.289). The interassay precision of amplification for the three days (n -- 30) was also calculated and found to be 0.5% for the target and 2.6% for the internal control.

B
--y = 35,73 + - 3 . 1 5 8 l o g ( x ) R= 0.9992 21.99 + -O,4011og(x) R= 0,9841

DISCUSSION
A new quantitative RT-PCR method, using the fluorescent hybridization probes and a sequence detector has been developed. This approach minimizes variable results caused by potential differences in the efficiency of reverse transcript i o n a n d a m p l i f i c a t i o n because b o t h target mRNA and internal control RNA are added to the same tube. Any amplification inhibitors present in a sample will affect amplification efficiency of both the target mRNA and the internal control RNA. Furthermore, only one set of primers is used to transcribe and amplify both target mRNA and internal control RNA, but different hybridization probes are used to ensure further the accuracy of the assay. Duplicate reactions, one containing the target mRNA probe and the other containing the internal control RNA probe, were prepared. This approach was used to enhance the dynamic range of the assay. Because specific hybridization of both primers and probe is necessary to generate a signal, the assay is both sensitive and specific. Amplification of DNA present in the total RNA preparation is avoided by using an exon/ intron junction s p a n n i n g forward primer, in which the 5' end of the primer is complementary to the 3' end sequences of one exon, and the 3' end of the primer complimentary to the 5' sequences of the next exon. Existing methods for quantitation of RT-PCR amplifications have been labor intensive, requiring each sample to be separated by gel electrophoresis, followed by quantitation of bands by ethidium bromide staining or isotopic labeling of the PCR products. One major advantage of the assay described above is the increased ease and

(,,)~e0B
o

18 = 17 16
I

Mean Cm C o m p e t i t o r

"~

- m - Mean Cm T a r g e t
~ ' ' ' ' ' r'k I

04

10 s

106

Relative Competitor Copies

Figure 3 QC RT-PCR. Duplicate sets of tubes containing fixed concentrations of adeno CFTR target were co-amplified with twofold serial dilutions of internal control RNA (n--3). Target hybridization probe was added to the first set of tubes, while internal control hybridization probe was added to the second set of tubes. Mean CT values of target and internal control are plotted against the relative copy number of internal control in each reaction tube. Target copy number is shown at the intersection of the two lines, and CT of target equals CT of internal control, and is multiplied by the respective target dilution to obtain the initial copy number for each microliter of target mRNA. (A) 1:25 dilution of target RNA; (B) 1:125 dilution of target RNA.

dilution of target and 1.2 · 108 copies of target/ ml for the 1:125 dilution of target, with a mean of 1.45 · 108 copies of target mRNA/ml stock solution.

lntra-assay and lnterassay Precision
To determine precision of the assay, 10 replicates 998 ~ GENOME RESEARCH

Downloaded from www.genome.org on August 26, 2007

Case 3:05-cv-04158-MHP

Document 187-12

Filed 08/29/2007

Page 7 of 9

A NOVEL METHOD FOR REAL TIME QUANTITATIVE RT-PCR

r a p i d i t y of s a m p l e a n a l y s i s , a l l o w i n g h i g h e r s a m p l e t h r o u g h p u t . By u s i n g real t i m e QC RTPCR, s a m p l e s can be reverse transcribed, a m p l i fied, a n d q u a n t i t a t e d i n one tube, w i t h o u t a n y f u r t h e r d o w n s t r e a m processing. C u r r e n t l y , u p to 96 reactions c a n be a n a l y z e d i n -3.5 h r u s i n g this assay format. A n o t h e r a d v a n t a g e is t h a t the CT value, used for q u a n t i t a t i o n , is m e a s u r e d d u r i n g a p e r i o d w h e n t h e PCR a m p l i f i c a t i o n is still i n the log p h a s e of a m p l i c o n a c c u m u l a t i o n . This circ u m v e n t s m a n y of t h e p r o b l e m s associated w i t h q u a n t i t a t i o n i n t h e p l a t e a u stage of a PCR a m p l i f i c a t i o n ( R a e y m a e k e r s 1995). In a d d i t i o n , t h e e l i m i n a t i o n of post-PCR s a m p l e m a n i p u l a t i o n s decreases t h e p o t e n t i a l of l a b o r a t o r y n u c l e i c acid c o n t a m i n a t i o n . F u r t h e r m o r e , well-to-well variations of fluorescence m e a s u r e m e n t can be norm a l i z e d to t h e q u e n c h i n g f l u o r e s c e n t d y e , TAMRA, b e c a u s e t h e e m i s s i o n i n t e n s i t y of TAMRA c h a n g e s very little d u r i n g t h e RT-PCR. C u r r e n t l y e a c h assay s a m p l e is a n a l y z e d in two different RT-PCR tubes: o n e c o n t a i n i n g the target probe a n d t h e o t h e r c o n t a i n i n g the internal c o n t r o l probe. U s i n g this assay, a d y n a m i c range of over 106s target m o l e c u l e s was o b t a i n e d . Assay t h r o u g h p u t c o u l d be i n c r e a s e d b y a d d i n g b o t h probes to t h e same RT-PCR tube. Reporter dyes w i t h less spectral overlap c o m p a r e d to FAM a n d TET m a y be useful for large d y n a m i c range assays i n a single tube. Precision of t h e QC RT-PCR assay was f o u n d to be excellent, w i t h intra-assay CVs of <2% a n d i n t e r a s s a y CVs of <3%. Q u a n t i t a t i o n of b o t h target a n d i n t e r n a l c o n t r o l were s h o w n to be l i n e a r over six logs a n d t h e assay c a n m e a s u r e as little as 1000 copies of m R N A per tube. Recent results indicate a m i n i m u m d e t e c t i o n a n d q u a n t i t a t i o n of 400 RNA m o l e c u l e s is possible (data n o t shown). A real t i m e PCR a s s a y u s i n g a c o n s t a n t a m o u n t of i n p u t DNA for each assay s a m p l e a n d a n o r m a l i z a t i o n gene to n o r m a l i z e for a n y m i n o r v a r i a t i o n s of i n p u t DNA c o n c e n t r a t i o n , has b e e n d e v e l o p e d (Held et al., this issue). Several q u a n titative RT-PCR assays w h i c h use h o u s e k e e p i n g genes as a n i n t e r n a l c o n t r o l h a v e also b e e n dev e l o p e d i n o u r l a b o r a t o r y (data n o t s h o w n ) . These a p p r o a c h e s e l i m i n a t e t h e n e e d to d e s i g n i n d i v i d u a l i n t e r n a l c o n t r o l t e m p l a t e s for e a c h gene i n a m u l t i g e n e e x p r e s s i o n assay. In c o n c l u s i o n , this m e t h o d for t h e q u a n t i t a t i o n of m R N A is sensitive, accurate, a n d can be used to q u a n t i t a t e large n u m b e r s of s a m p l e s in a relatively short time. The use of two d i f f e r e n t f l u o r e s c e n t h y b r i d i z a t i o n probes a n d a k n o w n

c o n c e n t r a t i o n of i n t e r n a l c o n t r o l RNA allows the initial m R N A c o p y n u m b e r of a n u n k n o w n target to be calculated.

METHODS Oligonucleotides
Table 1 shows the nucleotide sequences for the oligonucleotide hybridization probes and primers used. CFTR primers and probes were designed using the Oligo Version 4.0, National Biosciences, program. The forward primer (P1) was designed to span an exon/intron junction to avoid amplification of DNA sequences, whereas the reverse CFTR primer (P2) was complimentary to an exon. The CFTR target probe (prA) was labeled with FAM and the internal control probe (prB) with TET at the 5' end. Both probes were labeled with the quencher fluor TAMRA at the 3' end followed by the phosphorylation site p. Oligonucleotide hybridization probes (see Table 1) were obtained from Applied Biosystems Division, PerkinElmer (Foster City, CA). Primers were obtained from the Oligo Synthesis group, Genentech, Inc. (South San Francisco, CA).

Internal RNA Control
To construct the RNA internal control, a second set of primers (P3 and P4) were designed by appending the CFTR P1 and P2 primers to forward and reverse primers complementary to a pGEM-3Z plasmid (Clontech, Palo Alto, CA) sequence. The particular pGEM-3Z sequence was chosen because it contained similar G + C nucleotide content and was similar in size compared to the CFTR amplicon. Amplification of the pGEM-3Z plasmid with these primers yielded a DNA product consisting of an internal pGEM-3Z sequence with CFTR primer sequences at each 5' end [CFTR(P1)/pGEM-3Z/CFTR(P2)]. A third forward primer (P5) was constructed by appending the T7 phage promoter sequence, followed by an additional six bases (Stoflet et al. 1988), to the 5' end of the forward CFTR primer. Amplification of the CFTR(P1)/pGEM-3Z/CFTR(P2) DNA with the T7-containing forward CFTR primer (P5) and the reverse CFTR primer (P2) yielded an amplification product suitable for transcription, with the T7 promoter at the 5' end of the sense strand [T7/CFTR(P1)/pGEM-3Z/CFTR(P2)]. Each of the above PCR products was gel purified, using the QIAEX Gel Extraction Kit, (QIAGEN, Chatsworth, CA).

Transcription
Internal control DNA was transcribed using the T7 MEGAscript in vitro Transcription Kit (Ambion, Austin, TX) for large scale synthesis of RNA. Transcription was accomplished by following the Ambion protocol, and extending the incubation time at 37~ for 7 hr. After incubation the DNA was degraded by the addition of RNase-free DNase I, and the reaction was stopped, as described in the Ambion method. The RNA was recovered by extraction with phenol/CHC13, followed by precipitation of the RNAcontaining aqueous phase with one volume of isopropa-

GENOME RESEARCH@ 999

Downloaded from www.genome.org on August 26, 2007

Case 3:05-cv-04158-MHP
GIBSON ET AL.

Document 187-12

Filed 08/29/2007

Page 8 of 9

Table 1.

Primers and Probes
Base pairs 5'-CCGTGCCAAGAGTGACGTGTC-3' 5'-AAGCCAGCTCTCTATCCCA1-FCTC-3' 5'-CCGTGCCAAGAGTGACGTGTCCTATCGTCTTGAGTCCAACC-3' 21 24 41

Primer/probe
P1 P2 P3 CFTR primer, forward CFTR primer, reverse CFTR-pGEM-3Z primer forward CFTR-pGEM-3Z primer, reverse T7-CFTR primer, forward CFTR probe Competitor (pGFM-3Z) probe

P4

5'-AAG CCAGCTCTCTATCCCAI-ICTCATCCCI-IAACGTGAGT-I-FTC-3'

44

P5 pr6 pr7

5'-GGATCCTAATACGACTCACTATAGGGAGGCCGTGCCAAGAGTGA CGTGTC-3' 5' (FAM)-TGGACCAGACCAATTT1-GAGGAAAGGA-(TAMRA)p 3' 5' (TET)-TGGTATCTGCGCTCTGCTGAAGCC-(TAMRA)p 3'

50 27 24

nol. The RNA precipitate was w a s h e d two times in 75% ethanol, dried briefly in air, a n d r e s u s p e n d e d in RNase-free TE buffer (10 mM Tris-HCl at pH 7.6, 1 mM EDTA at pH 8.0). The c o n c e n t r a t i o n of t h e 319-bp internal c o n t r o l RNA was d e t e r m i n e d by ultraviolet spectroscopy.

Total RNA Extraction
RNAzol B (Tel-Test, Inc., Friendswood, TX) was used to extract total RNA from T84 cells infected with a recombinant, replication-deficient adenoviral vector c o n t a i n i n g the h u m a n CFTR cDNA. Cell m o n o l a y e r s were w a s h e d w i t h PBS, a n d 1 ml of RNAzol/lO 6 cells w i t h 4 units of RNase inhibitor (5 Prime-43 Prime, Inc., Boulder, CO) was added. The cells were solubilized by passing t h e lysate t h r o u g h t h e pipette a few times. O n e - t e n t h v o l u m e of c h l o r o f o r m was added, a n d t h e sample was c a p p e d a n d shaken vigorously, followed by a 5 m i n i n c u b a t i o n o n ice. The s u s p e n s i o n was centrifuged at 12,000g at 4~ for 15 m i n . The RNA in the aqueous phase was transferred to a clean tube. The RNA was precipitated w i t h an equal volu m e of isopropanol, stored for 15 m i n at 4~ a n d pelleted by centrifugation at 8000g for 8 m i n at 4~ The RNA pellet was w a s h e d twice w i t h 75% ethanol, air dried, a n d resusp e n d e d in TE w i t h 4 u n i t s / m l RNase inhibitor.

PCR
PCR reagents were o b t a i n e d from Boehringer M a n n h e i m (Indianapolis, IN). The following c o n d i t i o n s were used for PCR unless otherwise specified: Buffer c o m p o s e d of 10 mM Tris-HC1 (pH 8.3), 50 mM KC1, 1.5 mM MgC12, dNTPs at 0.2 mM, forward a n d reverse primers at 500 riM, a n d Taq polymerase at 5 U/IO0 ~L. Cycle parameters were 94~ for 2 rain, followed by 40 cycles of 94~ for 30 sec, 60~ for 30 sec, a n d 72~ for 1 rain, w i t h a final extension at 72~ for 7 min.

Cell Culture
H u m a n colon c a r c i n o m a T84 cells (ATCC CCL 248) were grown in a 1:1 m i x t u r e of Ham's F12 m e d i u m a n d Dulbecco's m o d i f i e d Eagle's m e d i u m w i t h 6% n e w b o r n calf ser u m (Gibco) a n d g e n t a m i c i n . For adenovirus infection, t h e cells were seeded in g r o w t h m e d i u m in 25 cc flasks a n d i n c u b a t e d o v e r n i g h t in a h u m i d i f i e d a t m o s p h e r e of 95% air/5% CO2 at 37~ The cells were w a s h e d with phosphate-buffered saline (PBS) a n d infected w i t h t h e replication-deficient a d e n o v i r u s c o n t a i n i n g the c o d i n g sequence of t h e CFTR gene at an a p p r o x i m a t e multiplicity of infection of 100 a n d i n c u b a t e d at 37~ overnight. The incubation m e d i u m was as described above, b u t with 2% of newb o r n calf serum. QC

RT-PCR

The Access RT-PCR System (Promega, Madison, WI) was used to reverse transcribe a n d amplify b o t h target a n d internal control. The reaction master m i x was prepared according to t h e m a n u f a c t u r e ' s protocol to give final concentrations of 1 · AMV/Tfl reaction buffer, 0.2 mM dNTPs, 1 mM MgSO4, 0.1 U / m l AMV Reverse Transcriptase, 0.1 U/l~l Tfl DNA Polymerase, a n d 250 nM CFTR primers P1 a n d P2. Target RNA (total RNA extracted from the adenovirus-infected cells) was a d d e d to t h e master mix. The master m i x was t h e n split into parts I a n d II a n d target hybridization probe was a d d e d to master m i x I, a n d internal control hybridization probe was a d d e d to master m i x II, to

1000 ~ GENOME RESEARCH

Downloaded from www.genome.org on August 26, 2007

Case 3:05-cv-04158-MHP

Document 187-12

Filed 08/29/2007

Page 9 of 9

A NOVEL METHOD FOR REAL TIME QUANTITATIVE RT-PCR
give final probe concentrations of 200 riM. Each master mix was transferred to a set of thermocycler tubes and twofold serial dilutions of internal control RNA were added to triplicate tubes of each master mix. Target and internal control were reverse transcribed at 48~ for 45 min, followed by 40 cycles of amplification at 95~ for 30 sec, 55~ for 45 sec, and 68~ for 1 rain, using the ATC. RT-PCR amplifications were also examined by agarose gel electrophoresis. After ethidium bromide staining, bands were visible only at the expected molecular weights for the CFTR mRNA and internal control products. Mullis, K., F. Faloona, S. Scharf, R. Saiki, G. Horn, and H. Erlich. 1986. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb. Symp. Quant. Biol. 51: 263-273. Piatak, M.J., K.C. Luk, B. Williams, and J.D. Lifson. 1993. Quantitative competitive polymerase chain reaction for accurate quantitation of HIV DNA and RNA species. BioTechniques 14: 70-81. Raeymaekers, L. 1995. A commentary on the practical applications of competitive PCR. Genome Res. 5" 91-94. Siebert, P.D. and J.W. Larrick. 1992. Competitive PCR. Nature 359: 557-558. Stoflet, E.S., D.D. Koeberl, G. Sarkar, and S.S. Sommer. 1988. Genomic amplification with transcript sequencing. Science 239: 491-494.

ACKNOWLEDGMENTS
We thank Ayly Tucker for the adeno CFTR primer design and the Genentech DNA Synthesis group for the primer synthesis. We also thank Junko Stevens, PE Applied Biosystems, for instructing us in the use of the ATC Model 7700 Sequence Detector, and R.G. Crystal, MD for the kind gift of AdGvCFTR.10 virus. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

Received June 19, 1996; accepted in revised form August 16, 1996.

REFERENCES
Becker-Andre, M. 1991. Quantitative evaluation of mRNA levels. Meth. Molec. Cell. Biol. 2: 189-201. Ferre, F. 1992. Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Applic. 2- 1-9. Gibbs, R.A. 1990. DNA amplification by the polymerase chain reaction. Anal. Chem. 62: 1202-1214. Heid, C., J. Stevens, K. Livak, and P.M. Williams. 1996. Real time quantitative PCR. Genome Res. (this issue). Holland, P.M., R.D. Abramson, R. Watson, and D.H. Gelfand. 1991. Detection of specific polymerase chain reaction product by utilizing the 5'--3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. 88: 7276-7280. Lee, L.G., C.R. Connell, and W. Bloch. 1993. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res. 21- 3761-3766. Livak, K.J., SO. Flood, J. Marmaro, W. Giusti, and K. Deetz. 1995a. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Applic. 4" 357-362. Livak, KJ., J. Marmaro, and J.A. Todd. 1995b. Towards fully automated genome-wide polymorphism screening. Nature Genet. 9" 341-342. McCulloch, R.K., C.S. Choong, and D.M. Hurley. 1995. An evaluation of type and size for use in the determination of mRNA by competitive PCR. PCR Methods Applic. 4: 219-226.

GENOME RESEARCH @ 1 O01