Improved RNase protection assay for quantifying LDL-receptor mRNA; Estimation of analytical imprecision and biological variance in peripheral blood mononuclear cells

N. E. Petersen*, L. K. Larsen, H. Nissen, Lillian Gryesten Jensen, A. Jensen, P. H. Petersen, M. Horder, N. Gregersen, K. Kristiansen

*Corresponding author for this work

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Abstract

We have improved the protocol for RNA quantification by using RNase protection. Instead of precipitation and extraction with phenol and chloroform, we use a taster and more reliable precipitation based on guanidinium thiocyanate (GdSCN). The internal standard is produced by in vitro transcription of a DNA template constructed so as to allow simultaneous detection of the in vitro transcript and the low-density lipoprotein receptor (LDLR) mRNA by use of the same probe and hybridization. Addition of this internal standard at the step for RNA isolation reduced the analytical imprecision from 40.8% to 19.3%. Estimates of the within- and between- subject biological variations of the LDLR mRNA content in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers were 21.5% and 13.6%, respectively, and the analytical imprecision was 22.6%. The mean content of LDLR mRNA in PBMCs from healthy individuals was 0.78 copies per cell.

Original languageEnglish
JournalClinical Chemistry
Volume41
Issue number11
Pages (from-to)1605-1613
Number of pages9
ISSN0009-9147
Publication statusPublished - 1. Jan 1995

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Keywords

  • internal standard to improve precision
  • variation, source of

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