Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization

Antonov, Janine; Goldstein, Darlene R; Oberli, Andrea; Baltzer, Anna; Pirotta, Marco; Fleischmann, Achim; Altermatt, Hans J; Jaggi, Rolf (2005). Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization. Laboratory investigation, 85(8), pp. 1040-50. New York, N.Y.: Nature Publishing Group 10.1038/labinvest.3700303

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Quantitative reverse transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate RNA abundance. The procedure is highly sensitive and reproducible as long as the initial RNA is intact. However, breaks in the RNA due to chemical or enzymatic cleavage may reduce the number of RNA molecules that contain intact amplicons. As a consequence, the number of molecules available for amplification decreases. We determined the relation between RNA fragmentation and threshold values (Ct values) in subsequent QRT-PCR for four genes in an experimental model of intact and partially hydrolyzed RNA derived from a cell line and we describe the relation between RNA integrity, amplicon size and Ct values in this biologically homogenous system. We demonstrate that degradation-related shifts of Ct values can be compensated by calculating delta Ct values between test genes and the mean values of several control genes. These delta Ct values are less sensitive to fragmentation of the RNA and are unaffected by varying amounts of input RNA. The feasibility of the procedure was demonstrated by comparing Ct values from a larger panel of genes in intact and in partially degraded RNA. We compared Ct values from intact RNA derived from well-preserved tumor material and from fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples of the same tumors. We demonstrate that the relative abundance of gene expression can be based on FFPE material even when the amount of RNA in the sample and the extent of fragmentation are not known.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Forschungsbereich Pathologie > Forschungsgruppe Molekularbiologie

UniBE Contributor:

Antonov, Janine; Oberli, Andrea; Baltzer, Anna and Jaggi, Rolf

ISSN:

0023-6837

Publisher:

Nature Publishing Group

Language:

English

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 15:12

Last Modified:

04 May 2014 23:23

Publisher DOI:

10.1038/labinvest.3700303

PubMed ID:

15951835

Web of Science ID:

000230645800011

URI:

https://boris.unibe.ch/id/eprint/31657 (FactScience: 196304)

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