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

N. E. Petersen*, L. K. Larsen, H. Nissen, Lillian Gryesten Jensen, A. Jensen, P. H. Petersen, M. Horder, N. Gregersen, K. Kristiansen

*Kontaktforfatter for dette arbejde

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

We have improved the protocol for RNA quantification by using RNase protection. Instead of precipitation and extraction with phenol and chloroform, we use a taster 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.

OriginalsprogEngelsk
TidsskriftClinical Chemistry
Vol/bind41
Udgave nummer11
Sider (fra-til)1605-1613
Antal sider9
ISSN0009-9147
StatusUdgivet - 1. jan. 1995

Fingeraftryk

LDL Receptors
Ribonucleases
Assays
Blood
Messenger RNA
RNA
Transcription
Chloroform
Phenol
DNA
In Vitro Techniques

Citer dette

Petersen, N. E. ; Larsen, L. K. ; Nissen, H. ; Jensen, Lillian Gryesten ; Jensen, A. ; Petersen, P. H. ; Horder, M. ; Gregersen, N. ; Kristiansen, K. / Improved RNase protection assay for quantifying LDL-receptor mRNA; Estimation of analytical imprecision and biological variance in peripheral blood mononuclear cells. I: Clinical Chemistry. 1995 ; Bind 41, Nr. 11. s. 1605-1613.
@article{1a3362b72ee244979c7d551a61bedb70,
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 taster 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.",
keywords = "internal standard to improve precision, variation, source of",
author = "Petersen, {N. E.} and Larsen, {L. K.} and H. Nissen and Jensen, {Lillian Gryesten} and A. Jensen and Petersen, {P. H.} and M. Horder and N. Gregersen and K. Kristiansen",
year = "1995",
month = "1",
day = "1",
language = "English",
volume = "41",
pages = "1605--1613",
journal = "Clinical Chemistry",
issn = "0009-9147",
publisher = "American Association for Clinical Chemistry, Inc.",
number = "11",

}

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, Lillian Gryesten; Jensen, A.; Petersen, P. H.; Horder, M.; Gregersen, N.; Kristiansen, K.

I: Clinical Chemistry, Bind 41, Nr. 11, 01.01.1995, s. 1605-1613.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

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, Lillian Gryesten

AU - Jensen, A.

AU - Petersen, P. H.

AU - Horder, M.

AU - Gregersen, N.

AU - Kristiansen, K.

PY - 1995/1/1

Y1 - 1995/1/1

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 taster 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 taster 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.

KW - internal standard to improve precision

KW - variation, source of

UR - http://www.scopus.com/inward/record.url?scp=0028807490&partnerID=8YFLogxK

M3 - Journal article

C2 - 7586550

AN - SCOPUS:0028807490

VL - 41

SP - 1605

EP - 1613

JO - Clinical Chemistry

JF - Clinical Chemistry

SN - 0009-9147

IS - 11

ER -