Ribopure rna isolation kit

A section of kidney cortex was snap‐frozen and stored at −80°C until use. RNA was extracted from frozen tissues using the RiboPure RNA isolation kit (Ambion, Austin, TX). RNA was reverse transcribed with random hexamer primers and the Superscript III kit (Invitrogen, Carlsbad, CA). Real‐time RT‐PCR was performed on a StepOne machine (Applied Biosystems, Mulgrave, VIC, Australia) with thermal cycling conditions of 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Primers and FAM‐labeled MGB probes for detection of CD68, TNF‐α, CCL2, KIM‐1, TGF‐β1, fibronectin, and collagen I and IV have been described previously (Tesch et al. 2015). Primers and VIC‐labeled probes for 18S ribosomal RNA were purchased from Applied Biosystems. All amplicons were normalized against the 18S RNA internal control (Applied Biosystems). The relative amount of individual mRNA species was calculated using the comparative Ct method. Real‐time PCR data were recorded as fold differences relative to nondiabetic females which was set to a value of 1.0.

Gene transcription was monitored with primers outlined in Supplementary Table 4. RNA extraction from bacteria was performed using the Ribopure RNA isolation kit (Invitrogen, Carlsbad, CA). Any remaining genomic DNA that may have been carried through the RNA extraction process was removed with a 1 h DNaseI treatment step, which was included in the kit. Following RNA extraction and DNaseI treatment, a DNA-based PCR was always performed to detect undigested DNA. The lack of amplification products confirmed the absence of contaminating genomic DNA. RT-PCR was carried out using the OneStep RT-PCR kit (Qiagen) according to the manufacturer’s recommendations. Briefly, each reaction setup consisted of 2 µl of dNTP mix (400 µM of each dNTP in a reaction), 0.6 µM of each primer, 2 µl of polymerase mix and 2 µl of specific RNA templates. The thermal cycling protocol was performed with the iCycler iQ multicolor real-time detection system (Bio-Rad, Hercules, CA) and contained an initial reverse transcription step of 50 °C for 30 min followed by PCR activation at 95 °C for 15 min. Subsequent steps in the protocol contained 40 cycles of amplification (94 °C for 45 s, 55 °C for 45 s, 72 °C for 1 min) and a concluding step at 72 °C for 10 min. Amplified products were analyzed on 1.8% (w/v) agarose gels stained with 0.5 µg/ml ethidium bromide and imaged with a VersaDocTM 4000 MP (BioRad).

Total RNA from cultured cells was extracted using RiboPure RNA isolation Kit according to manufacturer’s instructions (#AM1924, Invitrogen). Briefly, cells were homogenized in 1000μl of Tri-reagent (provided) and were then lysed with 200μl of chloroform. The sample was then vortexed and incubated at room temperature for 5 min followed by centrifugation at 12,000 x g for 10 min at 4°C to separate the mixture into a lower, red, organic phase, an interphase and a colorless, upper, aqueous phase. Upper aqueous phase was then collected and 200μl of ethanol was added. Each sample was then passed through the filter assembly resulting in the binding of the nucleic acids to the filter. The filter assembly was then rinsed with wash buffer and total RNA then eluted in a new tube for further analysis.
For detection of residual genomic and plasmid DNA, eluted RNA was subject to PCR reaction with primers specific to intron regions of house-keeping gene GAPDH, and with primers specific to plasmid transfected. Total RNA was converted to cDNA only if it is validated as free of DNA contamination.

The entire available adipose tissue sample (~50 to 100 mg) from each biopsy was used for total RNA extraction using the RiboPureTM RNA isolation kit (Ambion, Austin, TX, USA), following the manufacturer’s recommendations. The obtained RNA was quantified using a NanoDrop equipment (NanoDrop Technologies, Wilmington, DE, USA), and the RNA quality was assessed with an Agilent bioanalyzer device (Agilent Technologies, Palo Alto, CA, USA). The RNA integrity number values obtained for all the samples were in the range from 7.5 to 8.5. First-strand cDNA synthesis was carried out with Superscript II (Invitrogen Life Technologies, Paisley, UK) and random hexamers in a total volume of 20 μL containing 1 μg of total RNA, following the supplier’s instructions.

Total RNA was extracted from 50–100 mg of placental chorion obtained from 40 fetuses corresponding to both fetal genders and treatments (10 HTX-males, 10 HTX-females, 10 C-males and 10 C-females). RNA was extracted using RiboPureTM RNA isolation kit (Ambion, Austin, TX, USA) following the manufacturer’s recommendations. Samples were homogenized with 1 mL of TRI Reagent (Ambion, Austin, TX, USA), using an Ultra-Turrax® homogenizer and concentration and quality of the RNA obtained was determined using a NanoDrop equipment (NanoDrop Technologies, Wilmington, DE, USA) and an Agilent bioanalyzer device (Agilent Technologies, Palo Alto, CA, USA). First-strand cDNA synthesis was carried out with Superscript II (Invitrogen Life Technologies, Paisley, UK).
The qPCR assessment included genes involved in antioxidant homeostasis, vascularization and prenatal growth (SOD1, CAT, HIF1A, VEGFA, NOS2, IGF1 and UCP2) and it was performed as previously described for our group [50 (link)]. Stability of endogenous genes was tested with Genorm software [51 (link)] and finally, ACTB and B2M genes were employed for normalization. Data for primers (designed using Primer Select software; DNASTAR, Madison, WI, USA), amplicon lengths and PCR efficiencies are indicated in Table 6.

Muscle samples were maintained at −80 °C until gene expression analysis. For the transcriptome study, 36 muscle samples were used, corresponding to 12 Iberian animals belonging to the first slaughter (6 animals of each diet group) and 24 animals belonging to the second slaughter (12 animals of each breed, with 6 animals corresponding to each dietary group). The animals were randomly selected to perform transcriptomic analysis, representing all available litters. Total RNA was isolated from 50–100 mg samples of BF using the RiboPureTM RNA isolation kit (Ambion, Austin, TX, USA), following the manufacturer’s recommendations. The obtained RNA was quantified using NanoDrop equipment (NanoDrop Technologies, Wilmington, DE, USA), and the RNA quality was assessed with an Agilent 2100 bioanalyzer device (Agilent Technologies, Palo Alto, CA, USA) and submitted to the CNAG_CRG (Centro Nacional de Análisis Genómico, Barcelona, Spain). Libraries were prepared using the TruSeq mRNA-Seq sample preparation kit (Illumina Inc., Cat. # RS-100-0801, San Diego, CA, USA) according to manufacturer’s protocol. Each library was paired-end sequenced (2 × 75 bp) using TruSeq SBS Kit v3-HS, on a HiSeq2000 platform (Illumina, San Diego, CA, USA).

For the transcriptomic study, 24 animals were used (6 animals of each breed, corresponding to each diet group). Total RNA was isolated from 50–100-mg samples of subcutaneous ham fat using the RiboPureTM RNA isolation kit (Ambion, Austin, TX, USA), following the manufacturer’s recommendations. The obtained RNA was quantified using NanoDrop equipment (NanoDrop Technologies, Wilmington, DE, USA), and the RNA quality was assessed with an Agilent 2100 bioanalyzer device (Agilent Technologies, Palo Alto, CA, USA) and submitted to the Centro Nacional de Análisis Genómico (CNAG-CRG; Barcelona, Spain). Libraries were prepared using the TruSeq mRNA-Seq sample preparation kit (Illumina Inc., Cat. # RS-100-0801, San Diego, CA, USA) according to the manufacturer’s protocol. Each library was paired-end sequenced (2 × 75bp) by using TruSeq SBS Kit v3-HS in a HiSeq2000 platform (Illumina, Inc.).