Document Detail


Identification of stable endogenous reference genes for real-time PCR in the human fetal gonad using an external standard technique.
MedLine Citation:
PMID:  21527436     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Measurement of tissue mRNA transcript levels is critically dependent upon the normalization strategy used. For real-time polymerase chain reaction (PCR) (RT-PCR) this commonly depends upon identification of stable (non-variable) endogenous reference genes, with housekeeping genes (HKGs) the most commonly used. In this report we describe the use of an external mRNA standard to identify stable HKGs in the human fetal gonad. Total RNA was extracted from second trimester human fetal gonads and a standard amount of luciferase mRNA was added at the start of the extraction process. Levels of luciferase were then measured relative to each of seven HKGs (SDHA, TBP, B2M, PMM1, SFRS4, HMBS and UBC) by RT-PCR. When normalized to tissue weight, HKG expression was constant across fetal ages. Measurement of overall variation in transcript expression showed that PMM1 was the most stable HKG in the ovary while B2M was most stable in the testis. Re-analysis of the data using GeNorm and NormFinder algorithms showed that two of the top three most stable HKGs were the same using all three methods. This study describes a method for identification of endogenous, stable reference genes for RT-PCR studies of transcript expression levels which is objective and not dependent on prior assumptions of HKG expression. This technique is likely to be applicable to most tissues and, in this case, identifies suitable HKGs for studies into human gonadal development.
Authors:
Peter J O'Shaughnessy; Ana Monteiro; Paul A Fowler
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2011-04-28
Journal Detail:
Title:  Molecular human reproduction     Volume:  17     ISSN:  1460-2407     ISO Abbreviation:  Mol. Hum. Reprod.     Publication Date:  2011 Oct 
Date Detail:
Created Date:  2011-09-14     Completed Date:  2012-01-19     Revised Date:  2014-02-20    
Medline Journal Info:
Nlm Unique ID:  9513710     Medline TA:  Mol Hum Reprod     Country:  England    
Other Details:
Languages:  eng     Pagination:  620-5     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Female
Gene Expression Profiling / standards
Gene Expression Regulation, Developmental
Genes, Essential*
Gonads / embryology*
Humans
Luciferases / genetics
Male
RNA, Messenger / analysis*
Real-Time Polymerase Chain Reaction*
Grant Support
ID/Acronym/Agency:
CZB/4/742//Chief Scientist Office
Chemical
Reg. No./Substance:
0/RNA, Messenger; EC 1.13.12.-/Luciferases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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