Comparison of Methods for Quantitation of Radioactivity in Protected Hybrids in RNase Protection Assays

Several methods exist for the detection and quantitation of RNA, including Northern analysis, in situ hybridization, solution hybridization/RNase protection, dot blot and reverse transcriptase polymerase chain reaction (RT-PCR), and each has unique advantages. The solution-hybridization/RNase-protection assay is a highly sensitive and reproducible method of quantitating levels of specific mRNA transcripts. Its sensitivity offers the advantage of detecting low message levels and permits the use of relatively small amounts of starting tissue. Using the trichloroacetic (TCA) precipitation protocol described here, levels of mRNA lower than 1 pg can be accurately quantitated, allowing for the quantitation of mRNA at levels not detected by other means (9,12). The solution-hybridization/RNaseprotection assay uses a radiolabeled, antisense probe complementary to the RNA transcript to be measured. Total RNA isolated from tissues or cells is hybridized to excess radiolabeled probe, and the solution is treated with ribonucleases specific for singlestranded RNA, leaving double-stranded hybrids intact. Following RNase digestion, the most widely used protocol for measurement of protected hybrids involves gel electrophoresis followed by autoradiography and densitometry. An alternative method has been described in which hybrids are precipitated with TCA, filtered and analyzed by scintillation counting (1,3,4,6,7). The TCA precipitation method has several advantages. The protocol involves fewer steps and requires significantly less time to perform. A simplification of the experimental procedures reduces the number of steps in which human errors may be introduced. Finally, the ability to assay 100–300 samples in a single assay offers an advantage over gel electrophoresis, which is limited by the number of available wells in the gel; commercially available apparatuses are commonly limited to 40–60 samples. We conducted studies to compare the TCA precipitation method to the more widely used electrophoresis and autoradiography technique. Solutionhybridization/RNase-protection assays were analyzed in parallel by autoradiography with densitometry assessment and by TCA precipitation with scintillation counting. We also directly analyzed the agarose gels after autoradiography by excising bands from the gels for scintillation counting. Solution-hybridization/RNase-protection assays were carried out using probes for preproenkephalin (ppEnk) and proopiomelanocortin (POMC) mRNA. We measured ppEnk mRNA in total RNA samples isolated from the guinea pig caudate putamen, and we used the rat POMC probe to measure POMC mRNA from total RNA isolated from the rat anterior pituitary. All transcription templates were cloned in both sense and antisense orientations into the pSP64 or pSP65 plasmid vector (Promega, Madison, WI, USA). These vectors have the SP6 RNA transcription promoter located immediately 5′ to a multiple cloning site. The same promoter was used for transcription of both the antisense RNA riboprobes and sense transcript RNA that was used to construct the standard curves. The guinea pig ppEnk template was a 680-bp fragment from the coding region of the gene (8), and the rat POMC template was a 760-bp fragment from the cDNA (a generous gift of Dr. J. Roberts, Mount Sinai Medical Center). Plasmid DNA was isolated using alkaline lysis and centrifugation in cesium chloride, ethidium bromide equilibrium gradients (10). Transcription templates were prepared by digestion of plasmid DNA using HindIII for ppEnk and BamHI for POMC, cleaving 3′ with respect to the cloned fragment and leaving a 5′ overhang to prevent snap-back priming. Templates were then treated for 15 min at 37°C with 100 μg/mL proteinase K in the presence of 0.5% sodium dodecyl sulfate (SDS) to inactivate any ribonucleases present, followed by phenol extraction and ethanol precipitation. The radiolabeled probe was synthesized by the procedure of Melton et al. (11). Reaction conditions were as follows: 500 μM each of rCTP, rUTP and rATP, 8 μM rGTP, 1.25 μM [32P]GTP (3000 Ci/mmol; NEN Life Science Products, Boston, MA, USA), 0.5 μg of linearized plasmid, SP6 RNA polymerase (20 U; Promega), transcription buffer (40 mM Tris-HCl, pH 7.5, 6 mM MgCl2, 2 mM spermidine, 5 mM NaCl), 10 mM dithiothreitol (DTT) and RNase inhibitor (40 U; Promega) in a total volume of 20 μL. Nonradioactive GTP was added to increase the GTP concentration to reduce premature termination of the transcript (11). After a 1 h incubation at 37°C, 10 μg of tRNA (Type XX; Sigma Chemical, St. Louis, MO, USA) were added to aid RNA precipitation, and the plasmid was digested with 1 U of RNase-free DNase I (Promega) for an additional 15 min at 37°C. The reaction product was diluted in 1 mL TSE buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 7.5) and phenol-extracted. One volume of 100% ethanol was added to the aqueous phase to bring the solution to 50% ethanol. Single-stranded RNA was then purified by cellulose chromatography (5). One hundred thirty grams of CF11 cellulose (Whatman, Clifton, NJ, USA) were added to 1 L 0.1 M NaOH, washed 10 times with 1 L distilled (d)H2O and stored in 1 L 2 mM EDTA at room temperature. A Poly-Prep column (Bio-Rad, Hercules, CA, USA) was cast with a 0.2-mL bed of CF11, and the following washes were carried out: (i) 1 mL 50% ethanol, (ii) 1 mL TSE, (iii) 1 mL 50% ethanol, (iv) 8 mL dH2O and (v) 3 mL 50% ethanol. Following the final 50% ethanol wash, the RNA sample was loaded onto the column, and the flow-through was collected. The column was washed with 3 mL 35% ethanol, and RNA was subsequently eluted with 3× 0.9-mL aliquots of 22% ethanol and 3× 0.9-mL aliquots of dH2O. All ethanol solutions were prepared with TSE and degassed in a vacuum chamber before application to the column. The purification process was monitored by spotting 10-μL samples from each step (input, flowthrough, wash and each eluate) onto cellulose filter paper circles (Whatman), adding 4 mL liquid scintillation cocktail and measuring counts per