Improved RNase protection assay for quantifying LDL-receptor mRNA; estimation of analytical imprecision and biological variance in peripheral blood mononuclear cells
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Improved RNase protection assay for quantifying LDL-receptor mRNA; estimation of analytical imprecision and biological variance in peripheral blood mononuclear cells. / Petersen, N E; Larsen, L K; Nissen, H; Jensen, L G; Jensen, A; Hyltoft Petersen, P; Hørder; Gregersen, N; Kristiansen, K.
In: Clinical Chemistry, Vol. 41, No. 11, 1995, p. 1605-13.Research output: Contribution to journal › Journal article › peer-review
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TY - JOUR
T1 - Improved RNase protection assay for quantifying LDL-receptor mRNA; estimation of analytical imprecision and biological variance in peripheral blood mononuclear cells
AU - Petersen, N E
AU - Larsen, L K
AU - Nissen, H
AU - Jensen, L G
AU - Jensen, A
AU - Hyltoft Petersen, P
AU - Hørder, null
AU - Gregersen, N
AU - Kristiansen, K
N1 - 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
PY - 1995
Y1 - 1995
N2 - 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.
AB - 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.
M3 - Journal article
C2 - 7586550
VL - 41
SP - 1605
EP - 1613
JO - Clinical Chemistry
JF - Clinical Chemistry
SN - 0009-9147
IS - 11
ER -
ID: 11232020