Gotaq qpcr master mix reagent

We performed real-time qPCR in frozen renal tissue, assessing the following genes: VDR (Rn00690616_m1) and Klotho (Rn00580123_m1). We extracted and prepared total RNA by centrifugation technique using the commercial kit SV Total RNA Isolation System (Promega Corporation, WI). For cDNA synthesis, we used total RNA and GoTaq qPCR master mix reagent (Promega, Madison, WI). We performed real-time PCR using TaqMan (Applied Biosystems, Foster City, CA) on Step One Plus (Applied Biosystems). Primers were purchased from Applied Biosystems. We evaluated relative gene expression with the 2^−ΔΔCt method (17 (link)), using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the housekeeping gene (Rn01775763_g1).

Total RNA was isolated using TRI-reagent (Molecular Research Center Inc., Cincinnati, OH, USA) followed by LiCl purification. Precipitated and purified RNA was used for cDNA synthesis using Superscript II reverse transcriptase (Life Technologies, Thermo Fisher Scientific) according to the manufacturer's instructions. qPCR was done on a Rotorgene Q (Qiagen, Venlo, Netherlands) using GoTaq qPCR Mastermix reagent (Promega, Fitchburg, WI, USA). HH target genes were identified with primers as described in [16 (link)].
For Western blot analysis, proteins were visualized with horseradish peroxidase-conjugated secondary antibodies in combination with enhanced chemiluminescence detection system (GE Health Care, Chalfont St Giles, United Kingdom). The following antibodies were used: anti-GLI1 (V812; Cell Signaling Technology, Danvers, MA, USA), anti-GLI2 (H-300, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GLI3 (R&D Systems, Minneapolis, MN, USA), anti-DYRK1A, anti-DYRK1B (Cell Signaling Technology), anti-SUFU (C-15, Santa Cruz Biotechnology), anti-STAT3 (BD Biosciences, San Jose, CA, USA), anti-STAT5 (3H7, Cell Signaling Technology), anti-Beta Catenin (Cell Signaling Technology), anti-ACTB (Santa Cruz Biotechnology)

In silico prediction of putative GLI binding sites was done using the D‐Light Software29 (genome sequence GRCh37/hg19) trained with the GLI binding site matrix according to Winklmayr et al.30 The ENCyclopedia Of DNA Elements (ENCODE) Project31 was used to check for STAT3 binding regions. Luciferase reporter assays and site directed mutagenesis were carried out as described previously.23 All constructs were confirmed by sequencing.
Chromatin‐immunoprecipitation (ChIP) assays were carried out as described previously.32 A total of 10 μg cross‐linked chromatin was precipitated with antibodies listed in Supporting Information, Table S3. Immunoprecipitated DNA was analyzed by qPCR on a Rotor‐Gene Q (Qiagen) using GoTaq qPCR Master Mix reagent (Promega) with primers listed in Supporting Information, Table S2. The amount of immunoprecipitated DNA in each sample was calculated by the Percent Input Method according to the manufacturer's instructions (Cell Signaling Technology, Boston, MA).

We performed real-time qPCR in frozen renal tissue, assessing the following genes: VDR (Rn00690616_m1) and klotho (Rn00580123_m1). We extracted and prepared total RNA by centrifugation technique using the commercial kit SV Total RNA Isolation System (Promega Corporation, Madison, WI). For cDNA synthesis, we used total RNA and GoTaq qPCR master mix reagent (Promega, Madison, WI). We performed real-time PCR using TaqMan (Applied Biosystems, Foster City, CA) on Step One Plus (Applied Biosystems). Primers were purchased from Applied Biosystems. We evaluated relative gene expression with the 2^−ΔΔCt method (23 (link)), using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the housekeeping gene (Rn01775763_g1).

Total RNA was extracted from shoots of mutants and wild-type plants using TRIzol reagent (TaKaRa). RNA was used to synthesize cDNA using a PrimeScript™ RT reagent Kit from TaKaRa (code: RR047A). Q-PCR was performed on Bio-Rad CFX96 instrument with GoTaq qPCR Master Mix reagent (Promega) according to manufacturer’s instructions. The gene Actin1 as rice housekeeping gene was used for an internal reference. Each analysis includes three biological repeats and three technical replicates. Primers used for Q-PCR are listed in Additional file 5: Table S1.

The total RNA of the ARPE-19 cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA), and the cDNA was reverse transcribed using GoScript™ Reverse Transcription Mix (Promega Corporation). Gene expression was detected with GoTaq qPCR Master Mix reagents (Promega Corporation) in an ABI PRISM 7700 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The real-time PCR primer pairs were as follows: for GAPDH, 5′-CATCATCCCTGCCTCTACTG-3′ (forward) and 5′-GCCTGCTTCACCACCTTC-3′ (reverse); for BNIP3, 5′-GCCATCGGATTGGGGATCTAT-3′ (forward) and 5′-GCCACCCCAGGATCTAACAG-3′ (reverse); and for HIF-1α, 5′-GAACGTCGAAAAGAAAAGTCTCG-3′ (forward) and 5′-CCTTATCAAGATGCGAACTCACA-3’ (reverse). GAPDH was used as the internal control. All of the gene levels were normalized to the level of the GAPDH bene, and fold change was calculated by the 2^−ΔΔCt method.