Improved RNase protection assay for quantifying LDL-receptor mRNA; estimation of analytical imprecision and biological variance in peripheral blood mononuclear cells.
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.
Petersen
N E
NE
Department of Clinical Biochemistry, Odense University Hospital, Denmark.
Larsen
L K
LK
Nissen
H
H
Jensen
L G
LG
Jensen
A
A
Hyltoft Petersen
P
P
Hørder
Gregersen
N
N
Kristiansen
K
K
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
D006146:Guanidines; D015347:RNA Probes; D012333:RNA, Messenger; D011973:Receptors, LDL; D013861:Thiocyanates; C054436:guanidine thiocyanate; D012260:Ribonucleases