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© 1995 Oxford University Press 2947-2948

A homogeneous method to quantify mRNA levels: a hybridization of RNase protection and scintillation proximity assay technologies

A homogeneous method to quantify mRNA levels: a hybridization of RNase protection and scintillation proximity assay technologies Michael K. Kenrick, Lixin Jiang1, Cheryl L. Potts, Penny J. Owen, David J. Shuey1, Jason G. Econome1, John G. Anson* and Elaine M. Quinet1

Amersham International plc, Cardiff Laboratories, Forest Farm, Whitchurch, Cardiff CF4 7YT, UK and 1Wyeth-Ayerst Research, CN8000, Princeton, NJ 08543, USA

Received March 10, 1997; Revised and Accepted May 20, 1997

ABSTRACT

A novel method to measure mRNA levels has been developed by combining the detection capabilities of RNase protection (RPA) with the quantification advantages of scintillation proximity assay (SPA) technology. Sample processing is reduced to the addition of a single reagent post RNase digestion. As a model system, the inducible expression of rat apolipoprotein-A1 mRNA has been measured by both traditional gel-based RPAs and the SPA-based RPA assay. Results demonstrate that the ribonuclease protection proximity assay (RiPPA) faithfully reproduces the gel-based results and is at least as sensitive as many existing methods.

Ribonuclease protection (RPA) is generally regarded as being the most accurate and sensitive procedure which can provide specific mRNA mass data without target or signal amplification. The composite method described here, RiPPA, minimizes the labour and inherent error associated with existing techniques facilitating the rapid screening of large numbers of samples.

RiPPA utilizes a cRNA antisense probe which is dual-labelled with both radioisotope ([[alpha]-33P]UTP) and affinity recruitment (biotin) moieties. A molar excess of probe is hybridized to target mRNA and subsequently treated with RNase. Residual probe and non-complementary RNA are degraded. Resistant duplexes are recruited via the biotin moiety to streptavidin-coated SPA (1 ) fluoromicrospheres and the 33P-proximity signal is measured by scintillation counting (Fig. 1 ). The intensity of proximity signal is directly proportional to the amount of complementary RNA in the mixture.

This method was used to quantify the levels of apo-A1 mRNA in rodent livers. Apo-A1 is the major protein component of plasma high density lipoprotein (HDL). In transgenic mice, A-I over-expression was found to selectively increase HDL-cholesterol (HDL-C) levels (2 ). Human epidemiological studies demonstrate that apo-A1 is an important determinant of HDL-C levels and suggest that pharmacological interventions which increase apo-A1 production may raise HDL-C for the treatment of atherosclerosis (3 ). A selected agent was shown to increase HDL-C plasma apo-A1 and hepatic apo-A1 mRNA ~2-fold (E.M.Q., unpublished results). We have chosen this inducible system as a model for the validation of the RiPPA.


Figure 1.Schematic of RiPPA protocol. Total RNA (25 [mu]g) is dissolved in 30 [mu]l of 80% formamide, 40 mM PIPES, 0.4 M NaCl, 1 mM EDTA, pH 6.4 containing 2.5-3 * 105 c.p.m. of 33P-labelled RNA probe. After denaturation at 85oC for 5 min the mix was hybridized for 16-20 h at 48oC. Digestion buffer containing 50 mM sodium acetate, 0.1 M NaCl, 2 mM EDTA, pH 5.0, and RNase T2 (60 U/ml, Gibco-BRL) was added (350 [mu]l) and samples were incubated for 2 h at 37oC. The RiPPA was performed by taking the RNase-digested reactions and mixing with 1 ml of streptavidin-coated SPA beads (Amersham) freshly reconstituted to 0.5 mg/ml in assay buffer: phosphate-buffered saline, 0.1% (w/v) bovine serum albumin, 0.01% Triton X-100. After a 5 min incubation, the beads were pelleted and counted using a liquid scintillation counter.

Total RNA was isolated from the livers of six control and six drug-treated rats using the guanidinium thiocyanate method adapted for RNAzol (4 ). The riboprobe template consisted of a cDNA fragment spanning nt 94-317 of the rat apo-A1 sequence (5 ) subcloned into Bluescript SK(-). 33P-labelled antisense probes were synthesized by in vitro run-off transcription with T7 RNA polymerase. For RiPPA analysis, probes were dual-labelled by including Biotin-11-CTP (Amersham) into the reaction at a ratio of 10:1 (CTP:biotin-11-CTP). Sense-strand transcripts were generated with T3 RNA polymerase and were used for a target- dilution series to construct the standard curves.


Figure 2. Detection of specific mRNA species using the standard RPA procedure: gel analysis of protected fragments following hybridization and RNase treatment. 33P-radiolabelled antisense probes were hybridized with sense cRNA standards (lanes 1-5), ribosomal RNA (lane 6) and rat liver RNA samples (lanes 7-10) for 16 h. Following RNase digestion, organic extraction and precipitation, samples were analyzed by gel electrophoresis and quantified by PhosphorImager analysis. Lanes 1-5 contain sense cRNA apo-A1 standards (800, 400, 200, 100 and 50 pg respectively); lane 6 represents a negative control with 50 [mu]g of ribosomal RNA included; lanes 7 and 8 represent 25 [mu]g total liver RNA from control rats, and lanes 9 and 10 from drug-treated rats. The mass of RNA in each reaction tube is standardized to 50 [mu]g with ribosomal RNA or tRNA. A mouse GAPDH riboprobe is included as an internal standard (316 nt upper band) to normalize the relative abundance of apo-A1 transcripts (223 nt lower band).


Figure 3. RiPPA quantification of mRNA. (A) RiPPA standard curve; (B) correlation between gel-based RPA and RiPPA analysis of hepatic apo-A1 mRNA abundance. Values are means +- SEM, n = 6. *Significant difference between control and treated rats at P < 0.01.

Conventional RPA assays were performed as described previously (6 ) (Fig. 2 ). The labelled riboprobe protects fragments of the expected size for authentic apo-A1 mRNA (223 nt) and sense cRNA standards (296 nt). A 2-fold induction of apo-A1 mRNA levels is observed in drug-treated rats (Fig. 3 b).

Parallel assays were performed utilizing the RiPPA method. This method was developed and optimized with sense cRNA. A typical standard curve (Fig. 3 a) is shown. Extrapolation from this curve was used to determine the absolute amounts of specific apo-A1 transcripts. Significantly, the quantification and relative induction of apo-A1 mRNA levels as measured by RiPPA are concordant with the gel-based RPA determinations described above (Fig. 3 b).

The sensitivity of the RiPPA can be further improved by using riboprobes labelled with biotin and 33P to a specific activity in excess of 4 * 109 c.p.m./[mu]g according to the method of Harris et al. (7 ). No major increase in background was observed with these probes resulting in improved signal:noise ratios and a detection limit of <5 pg of specific target RNA. RNase A, requiring 0.375-0.5 M salt, was also used successfully in RiPPA (data not shown) as an alternative to RNase T2.

We have adapted the RiPPA into a 96-well format where the volumes of the hybridisation (20 [mu]l), RNase digestion (180 [mu]l) and SPA fluoromicrospheres (50 [mu]l) have been reduced (M.K.K., unpublished results). This assay was designed so that simple additions are made without the need for phenol extraction, ethanol precipitation and re-solubilization. RiPPA ensures rapid, more consistent results and confidence in statistical evaluation without sacrificing sensitivity. It is recommended that one confirms signal specificity by visualizing protected fragment sizes on gels prior to setting up a routine RiPPA regimen which should include positive and negative controls for RNase activity. Further improvements may be possible by reducing the hybridization time (8 ) and performing the RiPPA with direct cell lysates from defined cell culture populations.

ACKNOWLEDGEMENT

This work was performed as part of a collaboration between Amersham International plc and Wyeth-Ayerst Research.

REFERENCES

1 Bosworth,N. and Towers.P. (1989) Nature, 341, 167-168. MEDLINE Abstract

2 Rubin,E.M., Krauss,R.M., Spangler,E.A., Verstuyft,J.G. and Clift,S.M. (1991) Nature, 353, 265-267. MEDLINE Abstract

3 Gordon,D. and Rifkind,B. (1989) New Engl. J. Med., 321, 1311-1316. MEDLINE Abstract

4 Chomczynski,P. and Sacchi,N. (1987) Anal. Biochem., 162, 156-159. MEDLINE Abstract

5 Poncin,J.E., Martial,J.A. and Gielen,J.E. (1984) Eur. J. Biochem., 140, 493-498. MEDLINE Abstract

6 Quinet,E.M., Agellon,L.B., Kroon,P.A., Marcel,Y.L., Le,Y.-C., Whitlock,M.E. and Tall,A.R. (1990) J. Clin. Invest., 85, 357-363. MEDLINE Abstract

7 Harris,D.W., Kenrick,M.K., Pither,R.J., Anson,J.G. and Jones,D.A. (1996) Anal. Biochem., 243, 249-256. MEDLINE Abstract

8 Mironov,V.N., Van Montagu,M. and Inze,D. (1995) Nucleic Acids Res., 23, 3359-3360. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +44 1222 526409; Fax: +44 1222 526474; Email: john_anson@amersham.co.uk
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