RNA from kidney samples was isolated using the RNeasy kit (Roche, USA). Quantitative RT-PCR was carried out using an ABI PRISM 7300 Sequence Detection System. The final reaction contained template complementary DNA, iTaq SYBR Green Supermix with ROX (Bio-Rad, Hercules, CA, USA) and gene-specific primers. The sequences of the mouse primer pairs used in qRT-PCR are as follows: TGF-β1 (mouse) forward primer, 5′-TCGCTTTGTACAACAGCACC-3′, reverse primer, 5′-ACTGCTTCCCGAATGTCTGA-3′; HIF-1 a (mouse), forward primer, 5′-CTCACCAGACAGAGCAGGAA-3′, reverse primer, 5′-AAGGGAGCCATCATGTTCCA-3′; gp91-phox (mouse), forward primer, 5′-GAGGTTGGTTCGGTTTTGGC-3′, reverse primer, 5′-TGCACAGCAAAGTGATTGGC-3′; p67-phox (mouse), forward primer, 5′-AACATAGGCTGCGTGAACACTATCC-3′, reverse primer, 5′-GCAAGGTCGTACTTCTCCATTCTGTAG-3′. The conditions 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles for 30 s at 95 °C, 45 s at 60 °C, and 30s at 72 °C were applied. GADPH was used as an internal control. The cycle threshold values of GADPH and other specific genes were acquired after PCR. The normalized fold expression was obtained using the 2−ΔΔCT method. The results were expressed as the normalized fold expression for each gene.
Rneasy kit
Manufactured by Roche
Sourced in United States, Germany
The RNeasy kit is a product from Roche that is used for the isolation and purification of total RNA from various biological samples. It provides a simple and efficient method for extracting high-quality RNA for downstream applications such as RT-PCR, Northern blotting, and microarray analysis.
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2 protocols using rneasy kit
1
Kidney RNA Isolation and qRT-PCR Analysis
2
Transcriptomic Analysis of O. oeni
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O. oeni cells were harvested by centrifugation, frozen in liquid nitrogen and kept at -80ºC until RNA extraction. Total RNA extractions were performed using the Roche RNeasy kit following the manufacturer's instructions (Mannheim, Germany). RNA concentrations were calculated by measuring absorbance at 260 nm using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific SL, Alcobendas, Spain). cDNA was subsequently prepared. Samples were hybridized to the arrays for 18 hours at 42ºC. After washing, the arrays were scanned in a Roche Nimblegen MS 200 scanner. Raw data files (Pair and XYS files) were obtained from images using Nimblescan v2.6 software (Roche Nimblegen). Normalized gene expression values were obtained with Nimblescan software using the robust multichip average (RMA) algorithm as described by Irizarry et al. (2003a; 2003b) . Data univariate (ANOVA) analyses of transcriptomic data were conducted using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Variable means showing statistical significance were compared using Bonferroni post-test comparisons at a significance level of 0.05, after testing the homogeneity of variance assumption between the various groups. The results were submitted to GEO (Gene Expression Omnibus Database, NCBI) under accession number GSE62036.
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