Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 36, Num. 3, 2004, pp. 167-170

Indian Journal of Pharmacology, Vol. 36, No. 3, June, 2004, pp. 167-170

Research Paper

A semi-quantitative method for the estimation of adenosine A₁ receptor mRNA levels in rat kidney

Döndas N

Department of Pharmacology, Medical Faculty, Çukurova University, 01330, Adana
Correspondence Address:Department of Pharmacology, Medical Faculty, Çukurova University, 01330, Adana yakdas25@cu.edu.tr

Code Number: ph04056

ABSTRACT

INTRODUCTION

Adenosine is an endogenous nucleoside that modulates many physiological processes via its receptors including A₁, A_2a, A_2b and A₃.[1] However, a functional role has especially been ascribed to A₁ receptors in the kidney.[2] Therefore, adenosine A₁ receptors are extensively characterized in this tissue. But a sensitive method for the estimation of the mRNA levels of these receptors is still quite important, since the common methods including in situ hybridization, northern blot and slot blot analysis are no longer practical.[3] In the present study, an attempt has been made to develop a sensitive RT-PCR method for the detection of adenosine A₁ receptor mRNA levels in rat kidney.

MATERIAL AND METHODS

Experimental protocols
Small-scale total RNA isolation for the extraction and purification of total cellular RNA from whole rat kidney: 1) Male Wistar rats (200-250 g) were anesthetized with sodium thiobutabarbitone (180 mg/kg^-1, i.p.) and were killed by a blow to the head followed by exsanguination. Kidneys were removed and immediately freeze-clamped in liquid nitrogen and then stored at -70°C until required. 2) The kidney was homogenized in 10 ml of ice-cold denaturing solution (0.5 g/ml guanidium thiocyanate) with a Polytron homogeniser (2x15 second bursts). 3) 1.0 ml sodium acetate (2 M, pH 4.0) was added with mixing. 4) Five ml citrate-buffered phenol (pH 4.0) and 5 ml Chloroform-isoamyl alcohol (48:2 v/v) mix were added, mixed and chilled on ice for 15 min. 5) The mixture was then transferred to an eppendorf tube (1.5 ml) and centrifuged (13 krpm, 10 min), then the top aqueous phase was pipetted into a fresh tube. After the addition of an equal volume (10 ml) of isopropanol, the mixture was chilled at -20°C for 30 min. 6) The total cellular RNA was pelleted by centrifugation (13 krpm, 10 min). Then the supernatant was decanted and the pellet dried by inversion on a paper towel and it was resuspended in 500 ml denaturing solution with a brief heating at 65°C. 7) Total cellular RNA was again precipitated with 500 ml isopropanol and centrifugated (13 krpm, 10 min), then it was washed with 500 ml of ice-cold ethanol (70%) and re-centrifugated (13 krpm, 10 min). The pellet was dried at 37°C for 15 min and dissolved in 50 ml of DEPC (diethyl pyrocarbonate)-treated water with brief heating to 65°C to aid solubilisation. The resulting total cellular RNA was stored at -70°C.

Reverse transcriptase polymerase chain reaction (RT-PCR)

a) Reverse transcription (RT)
The concentration of total cellular RNA, isolated from whole rat kidney, was adjusted by UV spectrophotometry to 1 mg.ml^-1 in diethylpyrocarbonate-treated water. Then 2.0 ml of total RNA (1mg/ml) and 1.0 ml of oligo (dT)_12-18 primer (0.2 mg/ml) were added into a DEPC-treated eppendorf tube and heated at 65°C for 3 min and then the tube was cooled slowly to room temperature. After that 2 ml of DTT (dithiothreitol; 0.1M), 4 ml of 5x first strand buffer (250 mM Tris.Cl (pH 8.3), 375 mM KCl and 15 mM MgCl₂), 1 ml of dNTPs (deoxynucleoside triphosphates; 10 mM), 9 ml of RNA grade H₂O and 1 ml of Murine leukemia virus reverse transcriptase (MMLV RT; 200 U/ml) were added, mixed, and kept at 37°C for 15 min. Then the eppendorf tube was heated to 95°C for 3 min (to terminate the reaction and denature the mRNA). And then 5 ml of aliquot was taken and used as a template (complementary DNA; cDNA) in PCR.

b) Polymerase chain reaction (PCR)
Into a small eppendorf tube 5 ml of RT-template (cDNA), 5 ml of 10xPCR buffer (Tris.Cl [100 mM; pH 8.4], 0.5 M KCl, 1% Triton X-100), 4 ml MgCl₂ (25 mM), 1 ml of dNTPs (deoxynucleoside triphosphates; 2 mM ), 1 ml of N-terminus primer (5′-3′; 500 pmol), 1 ml of C-terminus primer (5′-3′; 500 pmol) (primer sequences were given in [Table - 1], 1ml of a-[³²P]-dCTP (2′-deoxycytidine-5′-triphosphate; 50 mCi, spec. act. 3000 Ci.mmol^-1 and 31 ml of MilliQ H₂O were added and mixed. Then 50 ml of mineral oil was added. After heating to 95°C for a few minutes, 1 ml of Taq (Thermophilus aquaticus) DNA polymerase (125 units) prepared according to the method of Pluthers (1993),[4] was added (The PCR protocol for adenosine A₁ receptors and b-actin are given in [Table - 2].

After thermocycling, 10 ml of PCR product was subjected to agarose gel (1% agarose gel and 0.1 mM ethidium bromide) electrophoresis [Figure - 1] and [Figure - 2]. To visualize the PCR product, the gel was dried overnight by sandwiching between 20-25 paper towels and applying a 1 kg weight. The dried gel was covered with cling film (Saran) and exposed to FujiBAS-IIIs imaging plate for 2 h prior to visualization on the FujiBAS 1000 bio-imaging analyzer. Then it was visualized and the intensity of the bands were quantitated.

Preparation and purification of cRNAs of adenosine

A₁ receptors and b-actin for the standard curve:
The cRNAs were produced by in vitro transcription of adenosine A₁ receptor and b-actin cDNAs in pSPORT2 and pBluescript II, respectively, with T7 RNA polymerase-based transcription kit. The protocol: 1) In a small 0.5 ml DEPC-treated eppendorf tube, 6 ml of RNase free H₂O, 2 ml of 10xBuffer, 2 ml of each nucleotide (ATP, GTP, CTP, UTP), 2 ml of template (bA or adenosine receptor cDNA; 1.0 mg/ml) and 2 ml of enzyme mix (T7 RNA polymerase and placental ribonuclease inhibitor) were added and incubated at 37°C for 4 h. 2). 1 ml RNA_ase free DNA_ase was added to the reaction and was incubated at 37°C for 15 min. 3). 115 ml of RNAase free H₂O and 15 ml of ammonium acetate were added to the reaction. Then it was cleaned by phenol/CHCl₃ extraction; 151 ml Phenol mix (Phenol:CHCl₃:Isoamylalcohol; 50:48:2 v/v) was added and centrifuged (13 krpm, 5 min). The top layer was taken into a fresh eppendorf tube and 151 ml CHCl₃ mix (CHCl₃:Isoamylalcohol; 48:2) was added. After mixing, the aliquot was centrifuged (13 krpm, 5 min). The top layer was taken into a fresh eppendorf tube, then 20 ml of sodium acetate (3M, pH 5.2) and 151 ml isopropanol were added and kept at -20°C for 30-60 min. After that the cRNA was pelleted by using a centrifuge (13 krpm, 10 min) and the supernatant was removed, then 500 ml of icecold ethanol (70%) was added. After centrifuging for 10 min at 13 krpm, ethanol was removed and the cRNA pellet was dried at 37°C for 10-15 min. Then it was dissolved in 10 ml water.

Preparation of standard curve
Standard curves were generated using varying amounts (1/3, 1/9, 1/27, 1/81, 1/243) of in vitro transcribed adenosine A₁ receptor and b-actin cRNAs. These serially diluted cRNAs were subjected to RT-PCR and the amount of the PCR product determined by densitometry. The band densities of the test samples were adjusted for concentration differences (dividing by the concentration in mg/ml) and then mRNA levels were extrapolated from the standard curve. To improve assay reproducibility, where numerous assays were performed at a time, a master-mix of all reagents was prepared and aliquoted to ensure that all assays received equal amounts and concentrations of substrate and reagents.

Materials
All oligonucleotide primers were obtained from Genosys Biotechnologies Ltd. Deoxyribonucleotides and oligo (dT)_12-18 primer were purchased from Pharmacia. M-MLV reverse transcriptase was obtained from Gibco-BRL (Life Technologies). a-[³²P] dCTP was obtained from ICN Pharmaceuticals. The Ambion mMESSAGE mMACHINE in vitro transcription kit was obtained from ams Biotechnology. MilliQ water (18 MW, Millipore) was used in the preparation of all reagents. For procedures involving RNA, all reagents and plasticware were treated with diethylpyrocarbonate (DEPC; 0.5%).

Analysis of data
mRNA levels were expressed as transcript number. Data are given as mean ± SEM and statistical comparison was made using t-test (One-Sample test). A value of P<0.05 was considered statistically significant. The intensities of the adenosine receptor mRNA bands were normalized relative to that of b-actin bands by dividing the former by the b-actin-specific PCR product densities. b-actin acted as a control for sample to sample variation in reverse transcription and PCR conditions, and the extent of degradation and recovery of RNA.[6]

RESULTS

The absolute mRNA levels of adenosine A₁ receptors in rat kidney were 1.30 ± 0.17 x 107 copies of adenosine A₁ receptor transcript/mg total RNA. The absolute mRNA levels of b-actin in the rat kidney were 2.26 ± 0.19 X 109 copies of b-actin transcript/mg total RNA. mRNA was extracted from the kidneys of six rats. b-actin or adenosine A₁ receptor mRNA levels in kidneys did not show statistically significant variations.

DISCUSSION

In the present study, a reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed and optimized to enable the absolute quantitation of the mRNA levels of adenosine A₁ receptors. Several techniques are currently available to measure changes in gene expression including Northern blot, RNase protection assay, in situ hybridisation, and RT-PCR. The Northern blot or the more sensitive RNase protection assay is sufficient to detect quantitative differences between samples. However, if the sample quantity is low or the target message is rare these techniques are no longer practical.[3] In the RT-PCR method, which is developed in the present study, less than 10 copies of target RNA can be estimated. This method is, actually, a semi-quantitative method.

In the present study, small-scale total RNA isolation protocol for the extraction and purification of total cellular RNA from the rat kidney was also developed. This is a slight modification of the method described by Chromoczynski and Sacchi (1987)[7] who used a large-scale total RNA isolation procedure. This modification was attempted since the new protocol of RNA isolation could be completed in half the time of the large-scale method, conducive to the handling of a large number of samples, and it used only a fraction of the reagents whilst maintaining the quality of the total cellular RNA isolated.

In conclusion, in the present study reverse transcriptase polymerase chain reaction (RT-PCR) technique was developed and optimized to quantitate the adenosine A₁ receptor mRNA levels. The precision of the method values indicates that the developed RT-PCR method for the estimation of adenosine A₁ receptor mRNA levels is a very sensitive, reliable and accurate one. Such a sensitive method is essential for the determination of extremely small amounts of mRNA levels.

ACKNOWLEDGEMENTS

I am grateful to Dr. C. J. Bowmer, Dr. A. Sivaprasadarao and Dr. M. J. Morton for their kind help.

REFERENCES

Copyright 2004 - Indian Journal of Pharmacology

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