Identifying the most suitable endogenous control for determining gene expression in hearts from organ donors.
BACKGROUND
Quantitative real-time reverse transcription PCR (qRT-PCR) is a useful tool for assessing gene expression in different tissues, but the choice of adequate controls is critical to normalise the results, thereby avoiding differences and maximizing sensitivity and accuracy. So far, many genes have been used as a single reference gene, without having previously verified their value as controls. This practice can lead to incorrect conclusions and recent evidence indicates a need to use the geometric mean of data from several control genes. Here, we identified an appropriate set of genes to be used as an endogenous reference for quantifying gene expression in human heart tissue.


RESULTS
Our findings indicate that out of ten commonly used reference genes (GADPH, PPIA, ACTB, YWHAZ, RRN18S, B2M, UBC, TBP, RPLP and HPRT), PPIA, RPLP and GADPH show the most stable gene transcription levels in left ventricle specimens obtained from organ donors, as assessed using geNorm and Normfinder software. The expression of TBP was found to be highly regulated.


CONCLUSIONS
We propose the use of PPIA, RPLP and GADPH as reference genes for the accurate normalisation of qRT-PCR performed on heart tissue. TBP should not be used as a control in this type of tissue.
Pérez
Silvia
S
Unidad de Coordinación de Trasplantes y Terapia Celular, Hospital Universitario Central de Asturias, C/Celestino Villamil s/n, 33006 Oviedo, Spain. silvia.perez@sespa.princast.es
Royo
Luis J
LJ
Astudillo
Aurora
A
Escudero
Dolores
D
Alvarez
Francisco
F
Rodríguez
Aida
A
Gómez
Enrique
E
Otero
Jesús
J
D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D020043:Genes, Essential; D006801:Humans; D009206:Myocardium / Q000378:metabolism*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012984:Software; D014019:Tissue Donors