PREPARATION AND CHARACTERIZATION OF RNA STANDARDS FOR USE IN QUANTITATIVE BRANCHED DNA HYBRIDIZATION ASSAYS

RNA standards were developed for use in quantitative hybridization ass ays such as the Quantiplex HCV RNA Assay and Quantiplex HIV RNA Assay, which are based on branched DNA signal amplification. In vitro transc ripts ranging in size from 0.5 to 9.4 kb were prepared and purified by phenol extraction following gel electrophoresis or column chromatogra phy. Aliquots of the transcripts were digested to nucleosides and phos phate and then quantified by phosphate analysis against the U.S. Natio nal Institute of Standards and Technology phosphate standard. The quan titation was checked by OD260 and by either hyperchromicity or isotopi c tracer analysis. The quantitation of each lot of RNA agreed within 2 0% by the three methods. The reproducibility of the methods was tested by preparing a total of 13 lots of standard RNAs. The average percent age full-length RNA of the 13 lots was 82%, with a range of 59 to 97%. The standard RNAs were used to test the ability of the branched DNA h ybridization assay to quantify all target RNAs accurately regardless o f size or slight variations in sequence. Standard Hepatitis C virus (H CV) RNAs of 1.3, 2.2, and 3.2 kb showed that size has no detectable ef fect on quantitation in the branched DNA hybridization assay. Three di fferent lots of standard 3.2-kb HCV RNA were serially diluted and quan tified over a thousand-fold range in the branched DNA hybridization as say. The average signal per attomole of target varied by less than 20% among the 3 lots. Standard HCV RNA transcripts were also prepared fro m clones of HCV subtypes 1b and 3a to study the effects of target sequ ence diversity and probe design on quantitation by hybridization. The quantitation of HCV subtype 1b RNA and 3a RNA differed by a factor of 1.6-fold in the branched DNA hybridization assay with one probe design and were indistinguishable with another probe design. (C) 1995 Academ ic Press, Inc.