Correcting false gene expression measurements from degraded RNA using RTQ-PCR

This paper describes a method allowing correcting false gene expression measured on highly degraded RNA using real-time quantitative reverse transcription-polymerase chain reaction (RTQ-PCR). RNA was isolated from different models (in vitro cell lines, in vivo models of human and dog) and different...

Full description

Saved in:

Bibliographic Details
Published in:	Diagnostic molecular pathology Vol. 16; no. 1; p. 38
Main Authors:	Port, Matthias, Schmelz, Hans Ulrich, Stassen, Tanja, Müeller, Kerstin, Stockinger, Marcus, Obermair, Richard, Abend, Michael
Format:	Journal Article
Language:	English
Published:	United States 01-03-2007
Subjects:	Animals Cell Line, Tumor Dogs Ethanol - chemistry Gene Expression Humans Kinetics Mice Neoplasms - chemistry Neoplasms - pathology Reverse Transcriptase Polymerase Chain Reaction - methods Ribonucleases - chemistry RNA Stability RNA, Neoplasm - analysis RNA, Neoplasm - standards Sensitivity and Specificity
Online Access:	Get more information
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	This paper describes a method allowing correcting false gene expression measured on highly degraded RNA using real-time quantitative reverse transcription-polymerase chain reaction (RTQ-PCR). RNA was isolated from different models (in vitro cell lines, in vivo models of human and dog) and different tissue types. In vitro RNA degradation and modeling of in vivo degradation were applied on intact and degraded total RNA. Gene expression (eg, Bcl-2, GAPDH, PGK, PSME3, RAB2, BAX) was measured using RTQ-PCR. 18S rRNA proved to be the most constant house-keeping gene. Less than 10-fold degraded RNA can be quantified correctly when using 18S rRNA for normalization purposes. Higher-fold degraded RNA can be quantified correctly up to a precision that is comparable to RTQ-PCR measurements on intact RNA when simulating the RNA-species and tissue-specific degradation kinetic.
ISSN:	1052-9551
DOI:	10.1097/01.pdm.0000213472.70054.94