Quantitative nuclear run-off transcription assay.

The nuclear run-off transcription assay is currently the most sensitive technique to measure the in situ transcription of specific genes. It provides information on the synthesis of a specific gene that occurs as a function of cell state and is unaffected by potentially confounding posttranscriptional events such as RNA processing, transport, editing, and mRNA degradation (2). Briefly, this assay takes advantage of the high incorporation rates of radiolabeled uridine 5′-triphosphates into both newly synthesized RNAs and those transcripts that already have been initiated. Characteristically, both nascent and elongated RNA molecules are detected by solid support techniques such as filter hybridization, in which in vitro synthesized transcripts bind to immobilized DNA oligonucleotides or templates (3). In principle, hybridization onto a solid support can be limited by the efficiency and specificity of the hybridization event. While alternative approaches such as RT-PCR have been suggested for the measurement of RNA transcripts (5,6), in the context of a nuclear run-off assay this technique may not be ideal because of the inability to account for inefficiencies in the PCR process along with varying fidelity and reproducibility. Significantly, the ribonuclease protection assay (RPA) as a solution hybridization method is more specific and 10–100 times more sensitive than filter hybridization, and, typically, less radioactive material is required. Moreover, RPA is associated with a wider linear relationship between mRNA amount and band intensity than solid support hybridization techniques, providing a method for RNA quantitation. The use of multiple probes also allows the simultaneous detection and measurement of several different transcripts, including an internal control from the same test-tube reaction (1,4,7). As applied to a nuclear run-off transcription assay, an RPA-based approach may also allow specific regions of the transcript to be investigated, which may be important in the investigation of very long genes. Here we have substituted RPA for filter hybridization as an alternative modification to the current protocol of nuclear run-off transcription assay. We were interested in the effect of mechanical force on vascular syndecan-4 gene expression in a rat pulmonary artery smooth muscle cell line (PAC-1). Cells at a density of 5 × 107/10-cm dish were plated on the flexible silicon membrane and subsequently exposed to cyclic strain at 10% strain amplitude and 1 Hz for 1 and 24 h. Cyclic strain is an in vitro mechanical stimulus used to mimic the circumferential deformation of the vessel wall in vivo. The cell monolayer was then extensively washed in DEPC-treated PBS and mechanically removed from the dish by sterile scrapers. Cells were harvested by centrifugation at 500× g for 5 min at 4°C, resuspended in 4 mL lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40), and thoroughly vortexmixed and incubated on ice for 10 min. A complete release of nuclei from cells was verified by a light microscope. Nuclei were collected by centrifugation at 500× g for 5 min at 4°C, washed in lysis buffer once, and suspended in 300 μL DEPC-treated water. An aliquot of this nuclear fraction was removed to determine the protein concentrations by BCA protein assay (Pierce Chemical, Rockford, IL, USA). For each sample, approximately 150 μg nuclear proteins were used for the further assay. The nuclear fraction was then mixed with an equal volume of 2× reaction buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 0.3 M KCl, 1 mM ATP, 1 mM CTP, 1 mM GTP, and 20 μM UTP), and transcription was initiated by the addition of 15 μL 10 mCi/mL [α-32P]UTP and 1 U/100 μL RNase inhibitor (6 U/sample; Roche Applied Science, Indianapolis, IN, USA). Incubation was carried out at 30°C for 2 h with gentle shaking. DNase I was prepared in 1 mL 10 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 50 mM MgCl2, 2 mM CaCl2 at a concentration of 40 μg/mL, and 0.6 mL of this solution were added to the reaction to terminate transcription. After a 10-min incubation at 30°C, protein digestion was initiated at 42°C for 30 min by the addition of 20 μL 5% SDS, 0.5 M Tris-HCl, pH 7.4, 0.125 M EDTA, and 10 μL 20 mg/mL proteinase K (200 μg/sample). Radiolabeled RNAs were sequentially extracted twice with an equal volume of ice-cold phenol:chloroform:isoamyl alBenchmarks