Predesigned primers

Predesigned primers (Sigma-Aldrich) were used for EvaGreen assays (S2 Table). Quantification of transcripts was carried out in a 15 μl reaction mix containing 1X SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA), 400 nM of each primer and 10 ng of purified WTA-cDNAs. The PCR data were collected using the CFX96 Real-Time System (Bio-Rad). Each sample was tested in duplicate. Calculation of normalized relative expression levels (fold change) and error propagation (standard error) was done using the Qbase Plus software version 3 (Biogazelle, Gent, Belgium). Normalization was performed using the two most stably expressed reference genes which were selected using the geNorm algorithm, among the following candidates: YWHAZ, GAPDH, HPRT1, UBC, B2M. A validation of results was performed (S1 Appendix).

Total RNA was extracted from whole kidney tissue using the RNeasy Plus Mini extraction kit (Qiagen Valencia, CA, USA). cDNA was synthesised using the Transcriptor First Strand cDNA synthesis kit (Roche Diagnostics, Mannheim, Germany). Predesigned primers (Sigma-Aldrich, NSW Australia) for fibronectin, collagen I and IV, αSMA, E-Cadherin and actin are listed in Table 2. Quantitative real-time PCR was performed with SensiMix SYBR hiRox (Bioline, NSW Australia) on the AB7900 machine (Applied Biosystems, Australia). Gene expression is presented as fold-change compared with control after normalisation to the housekeeping gene actin.Table 2

PCR primer sequences.

Target	Forward (5′-3′)	Reverse (5′-3′)
LOXL2	ATTAACCCCAACTATGAAGTG	CTGTCTCCTCACTGAAGGCTC
Fibronectin	ACAGAAATGACCATTGAAGG	TGTCTGGAGAAAGGTTGATT
COL1A1	CATGTTCAGCTTTGTGGACCT	GCAGCTGACTTCAGGGATGT
Col IV	TTAAAGGACTCCAGGGACCAC	CCCACTGAGCCCTGTCACAC
aSMA	ATAGGTGGTTTCGTGGATGC	ACTCTCTTCCAGCCATCTTTCA
E-Cadherin	CAAAGTGACGCTGAAGTCCA	TACACGCTGGGAAACATGAG
β-Actin	CTAAGGCCAACCGTGAAAAG	ACCAGAGGCATACAGGGACA