Validation of endogenous control genes for gene expression studies on human ocular surface epithelium

Kulkarni, Bina, Mohammed, Imran ORCID: https://orcid.org/0000-0002-8412-0768, Hopkinson, Andrew and Dua, Harminder S. 2011. Validation of endogenous control genes for gene expression studies on human ocular surface epithelium. PLoS ONE 6 (8) , 22301. 10.1371/journal.pone.0022301

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Abstract

Purpose To evaluate a panel of ten known endogenous control genes (ECG) with quantitative reverse transcription PCR (qPCR), for identification of stably expressed endogenous control genes in the ocular surface (OS) epithelial regions including cornea, limbus, limbal epithelial crypt and conjunctiva to normalise the quantitative reverse transcription PCR data of genes of interest expressed in above-mentioned regions. Method The lasermicrodissected (LMD) OS epithelial regions of cryosectioned corneoscleral buttons from the cadaver eyes were processed for RNA extraction and cDNA synthesis to detect genes of interest with qPCR. Gene expression of 10 known ECG—glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), peptidylprolyl isomerase (PPIA), TATA-box binding protein (TBP1), hypoxanthine guanine phosphoribosyl transferase (HPRT1), beta glucuronidase (GUSB), Eucaryotic 18S ribosomal RNA (18S), phosphoglycerate kinase (PGK1), beta-2-microglobulin (B2M), ribosomal protein, large, P0 (RPLP0)—was measured in the OS epithelial regions by qPCR method and the data collected was further analysed using geNorm software. Results The expression stability of ECGs in the OS epithelial regions in increasing order as determined with geNorm software is as follows: ACTB<18S

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Optometry and Vision Sciences
Additional Information:	This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication
Publisher:	Public Library of Science
ISSN:	1932-6203
Date of Acceptance:	21 June 2011
Last Modified:	07 Jun 2023 21:26
URI:	https://orca.cardiff.ac.uk/id/eprint/152693

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