Power pcr master mix 16

Total RNA was isolated from tissues using TRIsure reagent (BIOLINE, A Meridian life Science^® Company, WilfongRdMemphis, TN, United States) and 1 μg of RNA was reverse-transcribed by Quantitec reverse transcription kit (Qiagen, Milan, Italy) according to the manufacture’s instruction. The Q-PCR was performed with Power PCR Master Mix 16 (Applied Biosystems, Waltham, MA, United States) according to the manufacturer’s instruction and the following primers were used: Itgb1, CCAGCCAAGTGACATAGAGAA (forward) and GGTAATCTTCAGCCCTCTTGA (reverse); Aqp2, GCAGTT GTCACTGGCAAGT (forward) and AGGGGAACAGCAGGTA GTTG (reverse); V2r, GACTAAGTTGGCCTCCTGTGA (forward) and GGTCTCGGTCATCCAGTAGC (reverse); Gapdh, CTGTGGATGGCCCCTCTGGA (forward) and GGGCCCTCAGATGCCTGCTT (reverse). Reactions were run on a 7900HT system (Applied Biosystems, Waltham, MA, United States). GAPDH was used for normalization.

Total RNA was isolated from the kidney tissues using TRIsure reagent (BIOLINE, A Meridian life Science® Company, WilfongRdMemphis, TN, United States) and 1 μg of RNA was reverse- transcribed by Quantitec reverse transcription kit (Qiagen, Milan, Italy) according to the manufacture’s instruction. The q-PCR was performed with Power PCR Master Mix 16 (Applied Biosystems, Waltham, MA, United States) according to the manufacturer’s instruction and the primers listed in Table 1 were used. PCR setting was: denaturation step at 95 °C for 10 minutes, 40 cycles of denaturing at 95 °C for 10 s, annealing and extension at 60 °C for 1 minute then 95 °C for 15 s, 60 °C for 15 s and 95 °C for 15 s was performed on a 7900HT system (Applied Biosystems, Waltham, MA, United States). Data were analyzed using the ΔΔCt method, normalizing on GAPDH. Dissociation curves are showed in Supplementary Fig. S7.Table 1

List of primers used to perform q-PCR Table 1.

Gene	Sequence	Efficiency
Notch1 Forward	TCCCCTACAAGATCGAAGCC	0.84
Notch1 Reverse	CCCACAAAGAACAGGAGCAC
Notch2 Forward	TGTGGTCACTGATCCTTCCC	0.82
Notch2 Reverse	TCACATCCCTCCTCCCAAAC
Hes1 Forward	GTGGGTCCTAACGCAGTGTC	0.98
Hes1 Reverse	GTCAGAAGAGAGAGGTGGGCTA
Foxi1 Forward	CGTCTCACACTGAGCCAGAT	1.15
Foxi1 Reverse	TCACAGTTGGGGTCCAGAGT
NCC Forward	ATGTGGTACCCGCCTACGAA	1.11
NCC Reverse	GTGGCTACCTTCCTGCTTGA
GAPDH forward	CATGGCCTTCCGTGTTCCTA	0.94
GAPDH Reverse	CCTGCTTCACCACCTTCTTGA

One microgram of total RNA, isolated from tissues using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA), was reverse-transcribed by Quantitec reverse transcription kit (Qiagen) according to the manufacturer’s instructions.
The qPCR reaction mixture contained 7 ng of total cDNA, Power PCR Master Mix 16 (Applied Biosystems) and the following primers used at a concentration of 300 nm each:
Dicer1 Fw: TTGATGGGAACGCTAACACA; Rev: GCTCCAGGTTAAACCCATCA;
β-Catenin Fw: atggcttggaatgagactgc; Rev: atgctccatcatagggtcca;
RNA18SFw: CGGCTACCACATCCAAGGAA; Rev: GGGCCTCGAAAGAGTCCTGT.
Reactions were run on a 7900HT system (Applied Biosystems). For each sample, the expression of the genes of interest was normalized for the expression of RNA18S and measured under the same conditions.