title: Improved RNase protection assay for quantifying LDL-receptor mRNA; estimation of analytical imprecision and biological variance in peripheral blood mononuclear cells.
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 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.
authors.affiliation: Department of Clinical Biochemistry, Odense University Hospital, Denmark.
authors.forename: N E
authors.initials: NE
authors.lastname: Petersen
authors.forename: L K
authors.initials: LK
authors.lastname: Larsen
authors.forename: H
authors.initials: H
authors.lastname: Nissen
authors.forename: L G
authors.initials: LG
authors.lastname: Jensen
authors.forename: A
authors.initials: A
authors.lastname: Jensen
authors.forename: P
authors.initials: P
authors.lastname: Hyltoft Petersen
authors.lastname: Hørder
authors.forename: N
authors.initials: N
authors.lastname: Gregersen
authors.forename: K
authors.initials: K
authors.lastname: Kristiansen
mesh_terms: D011232:Chemical Precipitation; D006146:Guanidines; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D008099:Liver / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D015347:RNA Probes; D012333:RNA, Messenger / Q000097:blood*; D011973:Receptors, LDL / Q000235:genetics*; D012016:Reference Values; D015203:Reproducibility of Results; D012260:Ribonucleases; D012680:Sensitivity and Specificity; D013861:Thiocyanates
chemical_list: D006146:Guanidines; D015347:RNA Probes; D012333:RNA, Messenger; D011973:Receptors, LDL; D013861:Thiocyanates; C054436:guanidine thiocyanate; D012260:Ribonucleases