VALIDATION OF HOUSEKEEPER GENES FOR NORMALISATION FOR RT-qPCR GENE EXPRESSION STUDIES IN ISCHAEMIC AND TOXICOLOGICAL CONDITIONS IN RAT MODELS

S HERATH1, K TAYLOR1, A AU2, L SUCCAR1, Z  ENDRE2, J  ERLICH1
1University Of New South Wales , Kensington , Australia, 2Prince of Wales hospital, Randwick, Australia

Aim: To identify the most stably expressed housekeeper genes (HKG’s) under ischaemic and nephrotoxic conditions in various rat kidney models.
Background: Appropriate selection of housekeeper genes is pivotal to obtain accurate results in real time PCR. A number of studies have identified that the transcription levels of most commonly used HKG’s vary considerably under different experimental conditions.
Methods: Control and adenine fed Sprague Dawley (SD) rats were equally divided into 6 intervention groups each consisting of 8 biological replicates. The intervention groups were, subclinical CKD (sCKD), acute kidney injury (AKI) only, AKI on a background of sCKD, chronic kidney disease (CKD) and a control group. sCKD was induced with adenine and AKI was induced with cisplatin or ischaemia. 10 commonly used candidate HKG’s from different functional classes were selected. The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed utilizing NormFinder, Genorm, BestKeeper and comparative delta Ct statistical algorithms.
Results: Polyadenylate-binding nuclear protein 1 (PABPN1) and hydroxymethylbilane synthase (HMBS) were determined to be the most stable by all 4 algorithms. Bestkeeper/Genorm and Normfinder/comparative delta Ct approach respectively ranked succinate dehydrogenase (SDHA) and tyrosine-3-monooxygenase/tryptophan 5-monooxygenase-activation protein zeta (YWHAZ) as the third most stable HKG. A normalisation factor constructed by calculating the geometric mean of the three most stable HKG’s showed the least variability across the experimental groups. In comparison, the commonly used GAPDH and 18S were the least stable of all 10 HKG’s studied.
Conclusions: Construction of a normalisation factor by utilizing two or three most stable HKG’s which includes HMBS and PABPN1 produces the least variability to control for experimental errors and adjust for inter-sample variations in rat kidney studies.


Biography:
Fellow of the Royal Australasian College of Physicians and curently doing a PhD under the supervision of Professor Zoltan Endre and Dr Jonathan Erlich. Focus of current research is on ischaemia reperfusion injury especially in  the area of transplantation. Actively engaged in teaching at University of New South Wales and at Prince of Wales hospital, Randwick.

 

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