Document 0014
 DOCN  M9590014
 TI    Preparation and characterization of RNA standards for use in
       quantitative branched DNA hybridization assays.
 DT    9509
 AU    Collins ML; Zayati C; Detmer JJ; Daly B; Kolberg JA; Cha TA; Irvine BD;
       Tucker J; Urdea MS; Nucleic Acids Systems Department, Chiron
       Corporation, Emeryville,; California 94608, USA.
 SO    Anal Biochem. 1995 Mar 20;226(1):120-9. Unique Identifier : AIDSLINE
       MED/95305300
 AB    RNA standards were developed for use in quantitative hybridization
       assays such as the Quantiplex HCV RNA Assay and Quantiplex HIV RNA
       Assay, which are based on branched DNA signal amplification. In vitro
       transcripts ranging in size from 0.5 to 9.4 kb were prepared and
       purified by phenol extraction following gel electrophoresis or column
       chromatography. Aliquots of the transcripts were digested to nucleosides
       and phosphate and then quantified by phosphate analysis against the U.S.
       National Institute of Standards and Technology phosphate standard. The
       quantitation was checked by OD260 and by either hyperchromicity or
       isotopic tracer analysis. The quantitation of each lot of RNA agreed
       within 20% by the three methods. The reproducibility of the methods was
       tested by preparing a total of 13 lots of standard RNAs. The average
       percentage 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
       hybridization assay to quantify all target RNAs accurately regardless of
       size or slight variations in sequence. Standard Hepatitis C virus (HCV)
       RNAs of 1.3, 2.2, and 3.2 kb showed that size has no detectable effect
       on quantitation in the branched DNA hybridization assay. Three different
       lots of standard 3.2-kb HCV RNA were serially diluted and quantified
       over a thousand-fold range in the branched DNA hybridization assay. The
       average signal per attomole of target varied by less than 20% among the
       3 lots. Standard HCV RNA transcripts were also prepared from clones of
       HCV subtypes 1b and 3a to study the effects of target sequence diversity
       and probe design on quantitation by hybridization.(ABSTRACT TRUNCATED AT
       250 WORDS)
 DE    Base Sequence  DNA, Viral/*CHEMISTRY  Hepatitis C Viruses/GENETICS
       HIV/GENETICS  Molecular Sequence Data  *Nucleic Acid Hybridization
       Phosphates/ANALYSIS  Reference Standards  Reproducibility of Results
       RNA Probes/CHEMISTRY  RNA, Viral/CHEMISTRY/GENETICS/*STANDARDS
       Transcription, Genetic  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).