ANALYTICAL

BIOCHEMISTRY

196,%-94

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Quantitative Determination of mRNA Phenotypes by the Polymerase Chain Reaction Andrea

Ballagi-Pordany,*

And&

Ballagi-Pordany,?

and Keiko Funa*

*Ludwig Institute for Cancer Research, Biomedical Center, Box 595, S-751 24 Uppsala, The National Veterinary Institute, Biomedical Center, S-751 23 Uppsala, Sweden

Received

November

Inc.

The polymerase chain reaction (PCR)’ is a powerful method for amplifying specific DNA sequences in vitro, and its fields of application are growing very rapidly.

of

Virology,

Disadvantages of the method have been attributed to difficulties and unreliabilities with quantification of initial amounts of nucleic acids. Efforts have been made to improve reliability of quantification of PCR products by using labeled primers (1,2) as well as introduction of HPLC (3) and capillary electrophoresis (4). The possibility of phenotyping several mRNA species from a small number of cells by PCR was described by Rappole et al. (5,6). This method has been applied to the comparison of relative amounts of specific mRNA in different samples (7,8). Insufficient numbers of cells to be examined is a frequent problem in biological experiments and medical diagnostics, and it is therefore important to establish a method for quantification of several kinds of cellular RNA in a small number of cells. In this report, we describe a simple method for quantifying the amount of specific mRNA in different cells or tissue samples by using in vitro transcribed cRNA as a standard containing the same sequences as the corresponding mRNA. By this method, neither PCR with isotope-labeled primers nor polyacrylamide gel electrophoresis was applied, involving reagents which might be hazardous in a routine laboratory. A series of cRNA dilutions were reverse-transcribed, amplified, and dotblotted onto filter for hybridization. The intensity of the hybridization signals with a specific probe was correlated with the spectrophotometrically calculated amount of cRNA, and the regression line was used as a standard curve. The serial dilutions of total RNA of the samples were examined in the same manner and the copy number of the mRNA was calculated by fitting the hybridization results to the standard curve. MATERIALS RNA

’ Abbreviations used: PCR, polymerase chain reaction; PDGF, platelet-derived growth factor; CHO, Chinese hamster ovary; BSA, bovine serum albumin; AUC, area under curve. 0003-2697/91 Copyright All rights

and TDepartment

2, 1990

A method for quantitative determination of specific cellular mRNA is described. The mRNA in a dilution series of total RNA was reverse transcribed by an oligodT primer and the cDNA was amplified by the polymerase chain reaction (PCR) using sets of specific primers. A 32P- or biotin-labeled specific probe was hybridized to the PCR products immobilized on nitrocellulose membrane. The intensity of the hybridization signals was evaluated for quantification of the PCR products. A standard curve was produced by the known amount of the in vitro transcribed cRNA, which contained the same sequence as the mRNA. The series of standard cRNA dilutions were reverse transcribed, amplified and hybridized in the same manner. The amount of the specific RNA was deduced by fitting to the standard curve. Two tissue specimens of intestinal tumors, evaluated on the basis of hybridization signals by three different methods, were shown to contain similar amounts of @-actin mRNA. Furthermore, a Chinese hamster ovary (CHO) cell line transfected with platelet-derived growth factor (PDGF) @-receptor cDNA was found to contain similar amounts of @-actin mRNA as the untransfected CHO cell line. However, the transfected CHO cell line contained over 101’ copies of the PDGF P-receptor mRNA per microgram of total RNA, while the untransfected one showed no detectable RNA, indicating that the latter contained less than 10’ copies per microgram of total RNA in this assay. o 1991 Academic Press,

Sweden,

$3.00 0 1991 by Academic Press, of reproduction in any form

AND

METHODS

Preparation

The BamHI-cleaved cDNA for human P-actin (9), subcloned into SP65 vector (Promega Corp., Madison, 89

Inc. reserved.

90

BALLAGI-PORDhNY,

BALLAGI-PORDANY,

WI) and the P&I-cleaved cDNA for human PDGF @-receptor subcloned into pGEM vector (Promega) were used for in vitro transcription. The reaction was carried out using a kit (Promega) in a reaction volume of 100 ~1 following the vendor’s description. After incubation at 40°C for 1 h, the reaction was treated with RNase-free DNaseI (Promega) to remove the DNA template. The reaction mixture was extracted by phenol-chloroform and RNA was precipitated with ethanol, centrifuged and washed with 75% ethanol. The dried pellet was redissolved in 100 ~1 diethyl pyrocarbonate-treated water and the cRNA was quantified spectrophotometrically with absorbance at 260 nm. Then, 10 pg Escherichia coli tRNA and 50 U RNAguard (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) were added to protect specific cRNA. Total RNA from cell lines and tissue samples was obtained by the method of Chirgwin (10) using cesium chloride density centrifugation. Tissues from two intestinal carcinoid tumors were used for quantification of 8-actin mRNA. In addition, Chinese hamster ovary (CHO) cells transfected with a PDGF P-receptor cDNA (ll), as well as untransfected normal CHO cells were used for quantification of P-actin and PDGF p-receptor mRNA.

Reverse Transcription The cRNA solution was diluted lo-fold. We used dilutions containing lo”-10’ copies per 5 ~1 of the specific cRNA. The RNA of the samples was also diluted lo-fold before reverse transcription and amplification. The series of dilutions were reverse-transcribed into cDNA as previously described (12) with some modifications. Briefly, 5 ~1 of RNA solutions, which contained a known amount of RNA in the case of cRNA, were mixed with 200 U of M-MLV Reverse Transcriptase (BRL, Gaithersburg, MD), 10 mM of each dNTP (Pharmacia LKB Biotechnology AB), 0.2 pg oligo-dT primer (Pharmacia), and 20 U of RNAguard in PCR buffer, containing 10 mM Tris HCl, pH 8.3, 50 mM KCl, 0.1 mg/ml BSA, and 2.5 mM MgCl, in the final volume of 20 ~1. The solution was incubated at 37°C for 90 min, then heated up to 98°C for 10 min, and cooled down rapidly to 4°C.

PCR The nucleotide sequence of the upstream primer for @actin was AAG ATG ACC CAG ATC ATG TTT GAG, and that of the downstream primer AGG AGG AGC AAT GAT CTT GAT CTT. For PDGF P-receptor, the upstream primer was TCA ACG TCT CTG TGA ACG CAG TGC, and the downstream primer GCC CAG GGT GCG GTT GTC TTT GAA. Oligodeoxynucleotides were synthesized by a DNA synthesizer (Applied Biosystems, Model 380B, Foster City, CA) at the Ludwig Institute in Stockholm and purified through a Sephadex G-25 column.

AND

FUNA

The PCR was performed in an automatic DNA thermal cycler (Perkin-Elmer/Cetus, Norwalk, CT). Briefly, a reaction mixture contained the cDNA template, 0.4 pM of the upstream and downstream oligonucleotide primers, 0.2 mM deoxynucleotide triphosphates, and 1 U of the thermostable Taq DNA polymerase in the PCR buffer. The step-cycle program was set to denature at 94°C for 45 s, anneal at 55°C for 1 min, and extend at 72°C for 1 min for a total of 32 cycles.

Gel Electrophoresk

of the PCR Products

To determine the size of the PCR products, 10 ~1 of each product was run with molecular weight marker (HindIII-digested X DNA, Pharmacia) on 2.5% agarose gel at 100 V for 45 min and stained with ethidium bromide as described elsewhere (Fig. 1A) (13). The PCR products on the agarose gel were blotted onto a nitrocellulose filter and hybridized with a specific probe as described below to ascertain the specificity.

Probe-Labeling

for Hybridization

Probe sequences located between the two primers were chosen for hybridization with the PCR products so that only products with correct sequences were detected. Sequences having a sizeable homology with other sequences were avoided by screening with GenePro computer program (Riverside Scientific Enterprises, Bainbridge Island, WA). The nucleotide sequence of the probe for P-actin was AGG ATC TTC ATG AGG TAG TC. For the PDGF @-receptor, the probe sequence was TAG GTC CCC GAG TCT TCT AAC TC. The oligonucleotides were synthesized andpurified as described for the primers. The oligonucleotide probes were labeled with [32P]ATP (Amersham, Buckinghamshire, UK) by the 5’-end-labeling method, using T4 polynucleotide kinase (New England BioLabs, Beverly, MA) (14). The labeled probe was purified on a Sephadex G-50 column in a buffer containing 0.1 M Tris, pH 8.0,l mM EDTA, 0.1 M NaCl, and 0.5% SDS. Alternatively, the probes were labeled at the 3’-end by tailing with biotin-11-dUTP (Boehringer, Mannheim, Germany) using the terminal deoxynucleotidyl transferase enzyme (Pharmacia). The reaction was carried out for 2 h at 37°C as described elsewhere (15).

Hybridization The specificity and the quantity of the PCR products were analyzed by DNA dot-blot hybridization, as described elsewhere (14), with several modifications (16). Briefly, 10 ~1 of the PCR products was incubated at room temperature with 100 ~1 sodium hydroxide at a final concentration of 0.4 M in a microtiter plate. After 30 min the solutions were neutralized by 100 ~12 M am-

QUANTIFICATION

OF

mRNA

BY

THE

POLYMERASE Copy

Copy

numbers

of cRNA

prior

IO reverse

FIG. 1. A. Electrophoresis of PCR products on a reverse-transcribed lo-fold dilution 5-fold dilutions of the PCR products with 32P-labeled @-actin probe. The hybridized hybridization of the same PCR products with biotin-labeled o-actin probe.

monium acetate and &fold dilutions were prepared in 1 M ammonium acetate. The samples were transferred onto Hybond-C Extra nitrocellulose filters (Amersham) in a dot-blot apparatus (Bio-Rad, Richmond, CA). The filters were dried for 2 h at 80°C in a vacuum oven to immobilize the DNA. Prehybridization was performed at 43°C for at least 1 h and the filters were hybridized with the labeled probe at 43°C overnight. The temperature for the hybridization was calculated according to Davis et al. (14). The filters were washed in 2X SSC (1X SSC is 0.015 M sodium citrate and 0.15 M NaCl) once at 43°C for 15 min, twice in 2X SSC at room temperature for 30 min each. The hybridization conditions were identical for biotinylated and radioactive probes except for probe concentrations. For the biotinylated probes, 200 rig/ml final probe concentration and for the isotope-labeled probe, 2X lo6 cpm/ml were used. Quantification

of Detected Signals

Three filters of each dilution series were made to detect the signals using three different methods. Two filters were hybridized with the 32P-labeled probe and the radioactive signals were evaluated both by liquid scintillation counter and by densitometer after expo-

CHAIN numbers

of cRNA

91

REACTION prior

to reverse

transcription

transcription

series of @-actin cRNA. B. Dot-blot hybridization of the filter was exposed to X-ray film overnight. C. Dot-blot

sure to X-ray film. In brief, one filter was cut into small pieces corresponding to the DNA spots and measured in a Rack Beta liquid scintillation counter (LKB, Wallac, Finland) after addition of 5 ml OptiScint “Hi Safe” scintillation fluid (LKB), to each filter piece. The second filter was exposed overnight to an X-ray film (Hyperfilm-MP, Amersham). Intensity of the developed signals was analyzed by Shimadzu CS 930 densitometer (Shimadzu Corp., Kyoto, Japan) in the transmission mode. The third filter was hybridized with biotinylated probe and developed as described in the protocol of the Bethesda Research Laboratories (Gaithersburg, MD). The intensity of the color reaction products (dark blue) was quantified by the densitometer in the reflection mode. Logarithmically transformed data were analyzed by linear regression. We constructed standard curves from the data of cRNA dilution series, using the measurement of hybridization signals (cpm or AUC [area under the curve]) on the x axis (abscissa) and cRNA copy numbers calculated from the values obtained by spectrophotometry as they axis (ordinate). In this way, each of the three detection methods gave four standard curves according to the four 5-fold dilution steps in the microtiter

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log AUC FIG. 2. Regression curves produced by relating fi-actin cRNA copy numbers (r axis in ,,logarithmic scale) with the hybridization signals estimated by three different methods (r axis in ,,logarithmic scale). A. Scintillation counter measurement on hybridization signals. B. Densitometric analysis of hybridization signals expressed as AUC detected on X-ray film. C. Densitometric analysis of hybridization signals detected by biotinylated probe. The points between lo6 to 10” copy numbers with scintillation counter and 10’ to lo* with densitometric analysis of the signals on X-ray film were used to estimate regression curves. The amount of RNA in this range was considered to be measurable by this method. These are representative data of several experiments done on 5-fold diluted /3-actin cRNA.

plate before blotting the PCR products onto the nitrocellulose membranes (Figs. 1B and 1C). To estimate the specific mRNA copy numbers of the sample, we used the standard curves which yielded the most significant regression coefficients. In order to ascertain that the hybridization signals of the samples are in the linear phase of the curves, we made lo-fold dilutions on the total RNA of the samples before the reverse-transcription step. Following the amplification, the reaction solutions were treated in the same way as for the standards. Thus, we obtained similar patterns of the hybridization signals for the samples as in the case of cRNA, which were shown in Figs. 1B and 1C. The quantified signals were analyzed by linear regression and the regression coefficients were used to correct the measured values. The mRNA copy numbers of the samples were calculated by fitting these corrected data to the corresponding standard curve. RESULTS

The PCR products which were first analyzed by gel electrophoresis showed bands with the expected frag-

ment size, both for cRNA standards and the samples. The electrophoresis of PCR products on a reversed transcribed series of @actin cRNA dilutions is shown in Fig. 1A. When blotted to a filter, these bands hybridized with the specific probe (results not shown). For the quantification, the signal by dot-blot hybridization of PCR products was utilized as described above under Materials and Methods. Standard curves originating from the s-fold dilution series of PCR products demonstrated the widest ranges of linearity for both species of cRNA and were therefore applied for quantification (Figs. 2 and 3). In the cases of scintillation counting and biotin detection system, the linear ranges were seen when B-actin cRNA copy numbers were between lo6 to lOlo. Using densitometry on X-ray film, however, the linear range was narrower and found between 10’ to 10’ cRNA copy numbers. Table 1 shows the copy numbers of @-actin mRNA in two tumor samples based on the hybridization signals. The signals were detected by three different methods and the copy numbers were calculated by fitting to each standard curve generated by the cRNA. Independent of the de-

QUANTIFICATION 11

r

OF

mRNA

BY

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POLYMERASE

CHAIN

REACTION

93

TABLE

y-1.75+2.95x The @-Actin per 1 pg Total ceptor-Transfected

2

and PDGF P-Receptor RNA Estimated in CHO Cells

mRNA Copy Numbers Normal and PDGF P-R,-

RNA Source

of RNA

lo-fold PDGF

5-1.8

1.8

2

2.2

2.4

2.6

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Transfected CHO cells Normal CHO cells

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FIG. 3. Regression curve using PDGF P-receptor cRNA copy numbers as y axis (in Jogarithmic scale) and the hybridization signals measured by scintillation counter on hybridization signals as n axis (also in Jogarithmic scale). The points between lo6 to 10” copy numbers were used to construct the regression curve and the amount of RNA in this range was considered to be measurable by this method. These are representative data of several experiments.

tection methods, the specific copy numbers were in the same order of magnitude in both samples, between 0.57 and 3.85 X lo7 copy numbers/pg total RNA. The widest range was observed by densitometric analysis on X-ray film. When analyzing the different original dilutions of the samples prior to reverse transcription, the results were independent on the original dilutions when the detected signals were in the linear range of the standard curves. In this study, the lo-fold and loo-fold dilutions of the samples resulted in about the same copy numbers of specific mRNA as shown in Tables 1 and 2. We have also examined the CHO cell line transfected with PDGF P-receptor gene and compared it with the untransfected cell line for quantification of mRNA for PDGF P-receptor and /3-actin. For PDGF &receptor, hy-

TABLE

1

The @-A&in mRNA Copy Numbers per 1 pg Total RNA in Two Samples of Intestinal Tumor, Estimated by Three Different Detection Methods and Calculated by Fitting to the Corresponding Standard Curves Seen in Fig. 2 RNA

dilutions

lo-fold Sample

Sample

1

2

Scintillation counter X-ray film densitometry Biotin densitometry Scintillation counter X-ray film densitometry Biotin densitometry

Note. The lo-fold scription from RNA bers were calculated

1.63 x lo7 0.94 x 107 1.17 x 107 1.70 x 107 3.22 X 10’ 2.42 x lo7

tested loo-fold 1.58 X lo7 X lo7 x lo7 1.33 x lo7 0.57 x lo7 3.13 x lo7

3.85 3.30

and loo-fold dilutions made prior to reverse tranof the samples were examined and the copy numindependently.

P-receptor

dilutions

tested loo-fold

mRNA

1.00 x 10”