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

Research output: Contribution to journalJournal articleResearchpeer-review

  • N E Petersen
  • L K Larsen
  • H Nissen
  • L G Jensen
  • A Jensen
  • P Hyltoft Petersen
  • Hørder
  • N Gregersen
  • Kristiansen, Karsten
We have improved the protocol for RNA quantification by using RNase protection. Instead of precipitation and extraction with phenol and chloroform, we use a faster 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
Issue number11
Pages (from-to)1605-13
Number of pages8
Publication statusPublished - 1995
Externally publishedYes

Bibliographical note

Keywords: Guanidines; Humans; Leukocytes, Mononuclear; Liver; Nucleic Acid Hybridization; Precipitation; RNA Probes; RNA, Messenger; Receptors, LDL; Reference Values; Reproducibility of Results; Ribonucleases; Sensitivity and Specificity; Thiocyanates

ID: 11232020