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 journal › Journal article › Research › peer-review
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 language | English |
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Journal | Clinical Chemistry |
Volume | 41 |
Issue number | 11 |
Pages (from-to) | 1605-13 |
Number of pages | 8 |
ISSN | 0009-9147 |
Publication status | Published - 1995 |
Externally published | Yes |
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