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 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.
Port
Matthias
M
Bundeswehr Institute of Radiobiology, Munich, Germany.
Schmelz
Hans Ulrich
HU
Stassen
Tanja
T
Müeller
Kerstin
K
Stockinger
Marcus
M
Obermair
Richard
R
Abend
Michael
M
D000818:Animals; D045744:Cell Line, Tumor; D004285:Dogs; D000431:Ethanol / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D051379:Mice; D009369:Neoplasms / Q000737:chemistry / Q000473:pathology; D020871:RNA Stability; D012334:RNA, Neoplasm / Q000032:analysis* / Q000592:standards*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012260:Ribonucleases / Q000737:chemistry; D012680:Sensitivity and Specificity
D012334:RNA, Neoplasm; D000431:Ethanol; D012260:Ribonucleases