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Technical Briefs |
1
Department of Clinical Biochemistry, KH, Aarhus University Hospital, Nørrebrogade 44, DK-8000 Aarhus C, Denmark, and
2
Research Unit of Molecular Medicine, SKS, Aarhus University Hospital, 8200 Aarhus N, Denmark;
a author for correspondence: fax 45 8949 3060, e-mail domain31.ak14 s.akklkvt1{at}aaa.dk
Reverse transcription-PCR (RT-PCR), combined with an endogenous and exogenous standard, is used extensively to quantify specific mRNAs, but few studies have evaluated the reproducibility of these assays (1)(2)(3)(4)(5)(6)(7). Increased expression of the epidermal growth factor receptor (EGFr) has been observed in several human cancers and has been suggested to predict tumor progression (8)(9)(10)(11). Quantitative analysis for the EGFr mRNA using competitive RT-PCR has been described (12)(13)(14), but the analytical imprecision has not yet been documented systematically.
A primer pair was designed to flank the first intron of the human EGFr
gene (15)(16). The 350-bp cDNA fragment was
amplified as described below, using the following primers: primer I
(5'-GAC CCT CCG GGA CGG-3'), spanning positions 191–205; and primer II
(5'-GGC ATA GGA ATT TTC GTA GTA CAT AT-3'), spanning positions 515–540
and cloned into a pCRTM II vector (TA Cloning System,
Invitrogen). The "EGFr" plasmid was purified using a Qiagen plasmid
midi kit (Qiagen, Inc.) and cleaved by BglII (Pharmacia
Biotech). A 69-bp deletion was obtained by PCR (see below) using the
linear plasmid as template, primer III (5'-GAC CCT CCG GGA CGG CCG GGC
AAG GCA CGA GTA ACA AGC TCA CGC AGT TGG 3'), and primer II (Fig. 1
A). The PCR product was cloned into a pCR II vector,
purified, and cleaved as above. The PCR products of the EGFr and
standard were sequenced on both strands, confirming their identity and
the absence of PCR errors (data not shown).
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To synthesize the RNA standard, 5 µg of the linear plasmid (containing the 69-bp deletion) was incubated with 100 units of SP6 RNA polymerase according to the manufacturer's protocol (Promega Corp.). Fifty microliters of 25 mmol/L MgCl2 was added to the mixture before treatment with 20 U of RNase-free DNase I (Promega) for 30 min at 37 °C. The RNA standard was extracted using 1 volume of phenol:chloroform and centrifuged at 13 000g for 15 min at 4 °C. The aqueous phase was mixed with 1 volume of chloroform:isoamyl alcohol (49:1, by volume) and centrifuged as above. One volume of ethanol and 0.5 volume of 7.5 mol/L ammonium acetate were added to the aqueous phase. Precipitation was performed for 30 min at -80 °C, followed by centrifugation at 13 000g for 15 min at 4 °C. The pellet was resuspended in 40 µL of diethyl pyrocarbonate-treated double-distilled water (DEPC ddH2O). PCR was performed before and after the reverse transcriptase step. RNA concentration was determined by using an ultraviolet spectrophotometer (with absorbance at 260 nm of 1 corresponding to RNA = 40 mg/L) (17). RNA standards (3.7 x 10-8 and 3.7 x 10-12 mol/L) were stored in small aliquots at -150 °C and thawed a maximum of four times before disposal.
Human biopsies were obtained immediately after delivery from 12 healthy placentae in accordance with the Helsinki Declaration. From each placenta, four biopsies were removed, frozen immediately in liquid nitrogen, and stored at -150 °C.
EGFr protein was determined using an ELISA technique (18). Protein analysis was performed using a micro- titer assay (BCA protein assay reagent, Pierce).
Total RNA was extracted from 20 mg of placenta by adding 800 µL of a denaturing solution (solution D: 4 mol/L guanidine thiocyanate, 25 mmol/L sodium citrate, pH 7, 5 g/L sacrosyl, and 0.1 mmol/L 2-mercaptoethanol) and homogenized with a "turbo mixer" (Heidolph Diax 600) (19). The RNA pellet was resuspended in 25–40 µL DEPC ddH2O and stored at -150 °C. RNA (1:50 dilution in DEPC ddH2O) was quantified with use of an ultraviolet spectrophotometer (as above). For the imprecision experiments, RNAs from each placenta biopsy were pooled, and 20 aliquots were stored at -150 °C.
The reverse transcriptase reaction was performed as described in the manufacturer's protocol (Clontech 1 st. strandTM cDNA synthesis kit). Total RNA (0.5 µg) was converted to cDNA by priming with random hexamer primer (1 µmol/L) and MMLV reverse transcriptase (200 U/µg RNA) in a total volume of 20 µL. A 1:10 dilution was performed, and 10 µL cDNA was added to the PCR buffer containing 1x reaction buffer [10 mmol/L Tris-HCl (pH 9.0), 50 mmol/L KCl, 1.5 mmol/L MgCl2, and 0.01 g/L gelatin], 100 µmol/L deoxynucleotide triphosphates, 24.8 pmol primer I, 12.8 pmol primer II, and 1.0 U Taq DNA polymerase in a final volume of 50 µL (Pharmacia Biotech). A temperature cycling profile was designed (20). Each cycle consisted of denaturation at 94 °C for 30 s, annealing for 15 s, and extension at 74 °C for 1.5 min. Stepwise, the annealing temperature was decreased as follows: 2 cycles at 61 °C, 4 cycles at 60 °C, 6 cycles at 59 °C, and 28 cycles at 58 °C. The total cycle number was 40.
For competitive RT-PCR, a titration series of the RNA standard
was added to a constant amount of total sample RNA. The RT-PCR was
performed as described above. PCR products were analyzed by gel
electrophoresis (Fig. 1B
). Band density of the PCR products was
determined using a computerized analyzing system (Qgel, Quantigel
Corp.). Because of the presence of identical sequences in the EGFr cDNA
and standard cDNA, competitive amplifications of these two templates
led to hetero-duplex formation, as shown by denaturation/renaturation
of a mixture of EGFr and standard cDNAs. After densitometry, the target
and standard bands were corrected for hetero-duplex and size
differences (21)(22). The concentration of the
RNA standard and the ratio between standard and EGFr band density was
linear (Fig. 1
C); therefore, the EGFr concentration in the
sample corresponded to the standard concentration at the equivalence
point (ratio = 1).
Amplification kinetics of target and standard was evaluated by adding equal amounts of EGFr and standard linear plasmid (1 ng) to a single PCR mixture. After 25, 27, 30, 35, and 40 cycles, small portions of the PCR mixture were removed and analyzed by gel electrophoresis as above. The mean ratio between EGFr and the RNA standard was 1.03 ± 0.16 (mean ± SD, n = 5; data not shown), indicating that the amplification kinetics for the two fragments are fairly similar throughout the PCR.
To determine the imprecision of competitive RT-PCR for EGFr mRNA, competitive RT-PCR of pooled RNA was performed in duplicates on 19 separate days (23). Total and within-run CVs were 28% and 20% higher than the 10% CV reported for the EGFr protein assay (18).
The imprecision of the detection system was estimated on the basis of PCR products frozen in small portions. Gel electrophoresis and scanning were performed on 7 different days, analyzing five samples each day. Total and within-run CVs were 8% and 6% (23). Further optimization of the detection system will reduce the total analytical imprecision by <1%.
When analyzing biopsies, it is important to be aware of variation due to tissue inhomogeneity. The CV duplicates (23) for the mRNA concentrations determined in two biopsies from the same placenta (n = 12) was 38%. In two placentae, the variations between two sets of data (178 and 478 x 10-21 mol/µg RNA and 273 and 396 x 10-21 mol/µg RNA) were considerable. When these data were excluded, the CV was reduced to 20%. Based on our results, we recommend examining at least two biopsies from the same tissue and accepting data only if the difference between the samples meets predefined criteria for acceptance.
Only a few groups have reported on the imprecision of mRNA analysis using competitive PCR (1)(2)(3)(4)(6)(7)(24). Becker-André and Hahlbrock (25) were among the first to describe a competitive PCR assay by adding a standard (competitor) after the reverse transcriptase, allowing mRNA quantification by referring to a housekeeping enzyme mRNA. Two reports using this assay examined three different mRNAs and reported an imprecision between 14% and 27% (1)(3). Because the standard was added after the PCR, this variation reflects the PCR only. In competitive RT-PCR, the standard is added before the reverse transcriptase reaction (26). Ferre (24) developed a competitive RT-PCR analysis for HIV-I with a within-run analytical imprecision of 19%, which is comparable with the within-run imprecision we describe for the EGFr mRNA.
The amount of EGFr mRNA in the same samples was determined to be 170 (80–300) x 10-21 mol/µg RNA (mean and range, n = 10). The EGFr protein concentration in the placenta samples was 10.6 (7.4–14.4) x 10-12 mol/mg membrane protein (mean and range, n = 12). We did not find any significant correlation between EGFr mRNA and EGFr protein concentration in the placentae (Pearson r = 0.30, 2p = 0.40, n = 10) (data not shown). The lack of correlation may be explained by a combination of the narrow range covered for the EGFr, the high analytical imprecision for the mRNA analysis, or posttranscriptional regulation.
Acknowledgments
We thank Anna Lisa Christensen for excellent technical assistance. This work was supported by the Danish Cancer Society, Danish Research Council, Danish Research Academy, and Aarhus University Hospitals Research Initiative.
References
mRNA expression in breast cancer: evaluation of quantitative polymerase chain reaction for routine use. Br J Cancer 1996;73:1538-1544.
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mRNA expression. J Immunol Methods 1996;195:139-148.
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-carboxyglutamic acid protein mRNA measurement. Anal Biochem 1995;228:162-164.
[ISI][Medline]
[Order article via Infotrieve]
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X 174 RF
DNA/HaeIII digest); lanes 1–5, serial dilution
of standard and a constant amount of total RNA; lane 6,
control reaction without a template. (C) The gel was
subjected to digital scanning densitometry (Eagle Eye(TM),
Stratagene) and analyzed using a computerized analyzing system. The
target and standard were corrected by adding one-half of the
hetero-duplex band to each band. Because of size differences between
standard (281 bp) and target (350 bp), the standard was multiplied with
350/281. The RNA standard concentration per µg RNA was plotted
against the ratio of band densities of standard and target. The
equivalence point was determined by using linear regression and is
indicated by the arrow. Biopsies a (
) and b (
) from
the same placenta are shown.