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Assessing nuclear receptor isoforms using a standardized one‐step real time protocol (912.4)
Author(s) -
Sutherland Catherine,
Bistulfi Gaia
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.912.4
Subject(s) - gene isoform , amplicon , biology , computational biology , gene , complementary dna , nuclear receptor , function (biology) , gene expression , transcription factor , genetics , polymerase chain reaction
Nuclear receptors (NRs) are transcription factors able to regulate transcription based on environmental cues. They are fundamental for development and differentiation, and impaired NR function is associated with cancer and developmental diseases. There are more than fifty known NR families, each one comprising several genes encoding different isoforms. The relevance of NRs’ isoforms is unknown, but it is likely that they play a major role in modulating each other’s activity. The objective of this project was to develop an affordable, efficient, reliable method to quantify NR genes’ isoforms. We used one‐step real time PCR, which is more sensitive than the traditional two steps protocol, where retrotranscription of RNA into cDNA precedes real time analysis. To allow for absolute quantification of gene isoforms we recurred to a cRNA standard curve method, which accounts for different primer sets’ efficiencies. This method allowed us to compare expression levels of different amplicons specific to gene isoforms. Our results show that the method was quick, reliable and efficient in determining isoforms copy number, opening the way to studies focused at clarifying NRs’ isoforms function.