Rneasy lipid tissue midi kit

Adipose tissue SVFs were placed directly in RNA lysate buffer and RNA was extracted using the RNeasy kit (Qiagen). RNA was purified using RNeasy Lipid Tissue Midi Kit (Qiagen). Real-time PCR experiments were performed using QuantiTect SYBR green RT-PCR kit (Qiagen) using RNA samples with Corbett Rotor Gene 6000 Series (Qiagen, USA). Data analysis was performed by the comparative 2^^(−ddCT) method using Ct values.

Abdominal adipose RNA of male chickens (n = 6 per age group) was extracted with RNeasy Lipid Tissue Midi kit (Qiagen). RNA concentrations were measured with a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). All samples were analyzed with Experion RNA StdSens analysis kit to determine the quality. PCR primers were designed using Primer Express 2.0 (Applied Biosystems; see Table 1 for primer information). All RT-qPCR assays were conducted using QuantiTect SYBR Green RT-PCR kit (Qiagen). Reaction was done in 20 μL containing 50 ng of total RNA and 0.4 μM of each primer. Thermal cycles contained one cycle of pre-incubation at 50°C for 10 minutes and 95°C for 15 minutes, 35 cycles of amplification (95°C for 15 seconds and 60°C for 60 seconds). Primers were validated by melting curve analysis, standard curve, and nontemplate control reactions. For standard curve analysis, an RNA pool was made, serial-diluted to 0.08, 0.39, 1.56, 6.25, 25, 50, and 100 ng/μL, and measured again with spectrophotometer. Each concentration was analyzed in duplication with RT-qPCR to determine amplification efficiency.

Total RNA was extracted from the cortical and striatal Huntington’s disease and control brains (200–300 mg) using the RNeasy® Lipid Tissue Midi kit according to the manufacturer’s protocol (Qiagen). The RT-PCR was performed on 500 ng of RNA in a volume of 50 µl by using the Qiagen® Onestep RT-PCR according to standard procedures. The 4R and 3R MAPT transcripts were isolated using specific oligonucleotides (ForE9 5′-CTCCAAAATCAGGGGATCGC-3′, RevE12 5′-TTTTTATTTCCTCCGCCAG-3′) (Sigma) as described in Anfossi et al. (2011) (link). PCR products were separated by electrophoresis in 1% (w/v) agarose gel. The intensity of the bands was measured and the percentage of products corresponding to the 4R and 3R MAPT transcripts was calculated. For all quantitative experiments, RT-PCR reactions were carried out in three independent experiments. Statistical significance was ***P < 0.05, two-tailed t-test.

Tissue from the cortex of a 12 month-old cohort consisting of n = 3 per genotype for both male and female mice were collected for mRNA extraction. Whole-cortex RNA were isolated using the Qiagen RNeasy Lipid Tissue Midi Kit (Qiagen, Germantown, MD, USA; cat# 75144) according to the manufacturer’s instructions. Five hundred nanograms (500 ng) of RNA was then synthesized into cDNA by SuperScript II reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA; cat# 18064014) as per the manufacturer’s instructions. cDNA was then treated with RNase H (Thermo Fisher Scientific, cat# 18021071) for 20 min at 37 °C and stored at −20 °C until use. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using Power PCR SYBR Green Master Mix (Thermo Fisher Scientific, cat# 4368708) and the primers listed in Table 1. QRT-PCR was performed using a QuantStudio 6 Flex system (Applied Biosystems/Life Technologies, Waltham, MA, USA) and ∆∆Ct values were calculated using the housekeeping gene GAPDH as previously described [38 (link),44 (link)]. ANOVA analyses of male and female mice were performed independently. For visualization purposes and to illustrate the sex differential expression and detection, data were normalized to the male wildtype expression values of the gene.

Subjects were recruited by the endocrinology and surgery departments at the University Hospital Joan XXIII (Tarragona, Spain) in accordance with the Helsinki declaration. Human visceral and subcutaneous AT samples were obtained during surgery from lean and obese male and female individuals. Total RNA was extracted from adipose tissue using the RNeasy lipid tissue midi kit (Qiagen Science). One microgram of RNA was reverse transcribed with random primers using the reverse transcription system (Applied Biosystems) [39 (link)].
Mouse AT was obtained from wild type and Irs2^−/− [40 (link)] (insulin resistance and type 2 diabetes model) C57BL/6 littermates. According to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals”, all animals received humane care [22 (link)]. Total RNA was extracted from abdominal fat using a combined protocol including Trizol (Sigma) and RNeasy Mini Kit (Qiagen) with DNaseI Digestion. First-strand synthesis was performed using EcoDry Premix (Takara).

RNA extractions were performed using the RNeasy Lipid Tissue Midi Kit (Qiagen), which was designed for optimal lysis of tissue rich in lipids, following procedures previously optimised for use with human tissues [17 (link)]. The RNA concentration and quality were determined using a Nanodrop 2000 spectrophotometer and Agilent 2100 Bioanalyser respectively. Only RNA samples of excellent quality and integrity (RIN > 7) were used for gene expression and real-time RT-PCR analysis. The mean RIN values (± SEM) for each of the examined group of samples were 8.23 (1.88) for controls; 8.33 (0.72) for F+ GML; 7.79 (1.02) for F− GML; 8.36 (0.52) for F+ NAGM; 7.69 (1.09) for F− NAGM.

Total RNA was extracted from adipose tissue/cells using the RNeasy Lipid Tissue Midi Kit (Qiagen, Hilden, Germany). Total RNA quantity was measured at 260nm and purity was assessed by the OD260/OD280 ratio. One microgram of RNA was retrotranscribed with random primers using the Reverse Transcription System (Applied Biosystems, Foster City, CA, USA). Quantitative gene expression was evaluated by real-time PCR (qPCR) on a 7900HT Fast Real-Time PCR System using the TaqMan Gene Expression Assay (Applied Biosystems). The following genes were evaluated: ADRB1 (Hs 02330048_s1), ADRB2 (Hs 00240532_s1), ADRB3 (Hs 00609046_m1), GLUT1 (Hs 00892681_m1), GLUT3 (Hs 00359840_m1) and GLUT4 (Hs 00168966_m1). Results were calculated using the comparative Ct method (2-ΔΔCt), and expressed relative to the expression of the housekeeping genes cyclophilin 1A (PPIA) (Hs 04194521_s1) and 18S (Hs 03928985).