Convenient and Rapid Ribonuclease Protection Assay for Use with Primary Cell Cultures

The ribonuclease protection assay (RPA) is a commonly used method for the detection and quantitation of specific RNA species from total RNA preparations. This highly sensitive technique allows for the specific detection of target RNA species and is useful for evaluating large numbers of samples. Most standard RPA protocols recommend using 5–20 μg total RNA that is typically purified. However, when dealing with finite numbers of cells (i.e., primary myeloid leukocytes isolated from whole blood or small tissue sections) or using cells that have intrinsically low mRNA levels [i.e., lymphocytes or anucleated human platelets (4)], current RPA methods become limiting with regard to RNA yield. This aspect, accompanied with potential sample loss occurring through multiple RNA precipitation steps, limits the use of valuable RNA samples for other investigations, particularly if the sample is from an individual with a specific disease. We report here a simple modification of the ribonuclease protection assay that is especially useful for mRNA analysis of primary cell cultures where cell numbers and RNA yield are limiting factors. This convenient adaptation facilitates the rapid handling of large sample numbers through the elimination of multiple RNA precipitation steps without sacrificing the detection of multiple RNA species within a single sample and is of use when assaying changes in transcript levels based on a constant number of cells at a specific starting point (i.e., time-course analysis). To illustrate the effectiveness of this modified rapid protocol as compared to standard RPA methods, the levels of constitutively expressed RNA species commonly used as internal standards were evaluated in primary human cell types. Human peripheral monocytes were isolated from healthy donors and purified from whole blood by countercurrent elutriation (5). Confluent primary cultures of human umbilical vein endothelial cells (HUVEC) were established in 0.2% gelatin-coated six-well dishes as previously described (6). Human platelets were isolated from whole blood as previously described (5). Cell disruption and RNA isolation from 1 × 106 monocytes, 1 × 106 HUVEC, and 1 × 108 platelets was accomplished using 1 mL TRIZOL® (Invitrogen, Carlsbad, CA, USA). In the standard RPA method, total RNA is precipitated out of the aqueous phase with isopropanol, brought up into double-distilled water treated with 0.1% DEPC, and quantitated to determine the yield. The typical yield of total RNA using these cell isolation conditions was 5.8 ± 0.5 μg from 1 × 106 monocytes, 10.5 ± 0.9 μg from 1 × 106 HUVEC, and 6.2 ± 0.5 μg from 1 × 108 platelets. Both the sample RNA and low specific activity antisense riboprobes for β-actin, GAPDH, and 28S RNAs were co-precipitated with ethanol as described (1). With the rapid RPA method, riboprobes are directly added to the extracted RNA-containing aqueous phase, and both the sample RNA and probes were co-precipitated with isopropanol; this allows for the quantitative recovery of cellular RNA to be monitored. To maintain equal amounts of RNA between reactions, a fixed volume of aqueous phase (500 Benchmarks