From human adipose tissue, total mRNA for gene expression was extracted by the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) and for methylation studies, DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The concentration of mRNA was determined with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). The mRNA integrity was analyzed by an Experion Automated Electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA). From rat inguinal (subcutaneous) adipose tissue, total RNA was isolated by the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) and cDNA was prepared using the High Capacity RNA to DNA kit (Applied Biosystems, Hercules, CA, USA).
Rneasy lipid tissue mini kit
Manufactured by Qiagen
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The RNeasy Lipid Tissue Mini Kit is a laboratory equipment product designed for the purification of total RNA from lipid-rich tissues. It is used to extract high-quality RNA from samples such as adipose tissue, brain, and other lipid-containing tissues.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (145 mentions)
Nanodrop, by Thermo Fisher Scientific (83 mentions)
Qiazol lysis reagent, by Qiagen (74 mentions)
Rneasy mini kit, by Qiagen (67 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (61 mentions)
Iscript cdna synthesis kit, by Bio-Rad (58 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (55 mentions)
Quantitect reverse transcription kit, by Qiagen (51 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (49 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (46 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (43 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (42 mentions)
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2100 bioanalyzer, by Agilent Technologies (37 mentions)
Rnase free dnase set, by Qiagen (33 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (33 mentions)
Trizol reagent, by Thermo Fisher Scientific (32 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (30 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (27 mentions)
1 272 protocols using rneasy lipid tissue mini kit
1
Tissue-Specific RNA and DNA Extraction
2
Arthritis and Brain RNA Expression Analysis
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For arthritis analysis, RNA extraction and real-time polymerase chain reaction (RT-PCR) were performed as previously described [21 (link)]. Briefly, total RNA was extracted from four amputated limbs using a RNeasy Lipid Tissue Mini Kit (Qiagen, Tokyo, Japan). Real-time PCR was performed using an Applied Biosystem StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) with Taqman probes and the following primers: IL-1β (Mm01336189_m1), IL-6 (Mm00446190_m1), and Actb (Mm00607939_s1).
For brain analysis, RNA extraction and RT-PCR were performed as follows. After transcardial perfusion with PBS, total RNA was extracted from the dissected medulla containing of the AP (from the rostral to caudal end) using a RNeasy Lipid Tissue Mini Kit (Qiagen). mRNA extraction from the whole brain after separating the medulla oblongata was similarly performed. RT-PCR was performed as described above with the following primers: IL-1β (Mm01336189_m1), IL-6 (Mm00446190_m1), TNF-α (Mm00443258_m1), TGF-β (Mm01178820_m1), Itgam (Mm00434455_m1), and Gapdh (Mm99999915_ g1).
Expression levels normalized to Actb or Gapdh were analyzed using the ΔΔCT method. mRNA expression levels were represented as values relative to the average of the saline group.
For brain analysis, RNA extraction and RT-PCR were performed as follows. After transcardial perfusion with PBS, total RNA was extracted from the dissected medulla containing of the AP (from the rostral to caudal end) using a RNeasy Lipid Tissue Mini Kit (Qiagen). mRNA extraction from the whole brain after separating the medulla oblongata was similarly performed. RT-PCR was performed as described above with the following primers: IL-1β (Mm01336189_m1), IL-6 (Mm00446190_m1), TNF-α (Mm00443258_m1), TGF-β (Mm01178820_m1), Itgam (Mm00434455_m1), and Gapdh (Mm99999915_ g1).
Expression levels normalized to Actb or Gapdh were analyzed using the ΔΔCT method. mRNA expression levels were represented as values relative to the average of the saline group.
3
RNA Extraction from Adipose Tissue
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Isolated adipocytes and stromal vascular cells snap frozen in trizol were thawed on ice and then transferred to Fastprep matrix D lysing tubes (Mp Biomedicals) for homogenization using a Qialyzer instrument (Qiagen). Cells were homogenized with 2 cycles of 2 minutes at 20 1/s frequency, rotating tubes within holders (outside to inside and vice-versa) between rounds. RNA was extracted from the tissue homogenates using the RNeasy Lipid Tissue Mini kit and the Qiagen user-developed protocol titled Purification of total RNA from fatty tissues using the RNeasy Lipid Tissue Mini kit and MaXtract High Density (Qiagen). On-column DNA digest was performed for all samples according to manufacturer protocol (Qiagen). RNA was quantified using a Nanodrop 8000 spectrophotometer and quality was assessed using an Agilent 4100 Bioanalyzer (Agilent). cDNA was synthesized from RNA using High-capacity cDNA reverse transcription kits (Applied Biosystems).
4
Isolation and Characterization of Biomolecules
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Total RNA from, liver, lung and testicle was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany) and RNA from hypothalamus and adipose tissue was obtained using RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. This procedure allows the isolation of total RNA, DNA and protein fractions from a single sample44 .
Complementary DNA (cDNA) was synthesized from 1 μg of DNase-treated RNA45 (link). Protein pellets obtained using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany),were resuspended in 4% SDS and 8 M urea in 40 mM Tris–HCl. The total recovery and integrity of these fractions were determined by Lowry et al.46 (link).
Complementary DNA (cDNA) was synthesized from 1 μg of DNase-treated RNA45 (link). Protein pellets obtained using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany),were resuspended in 4% SDS and 8 M urea in 40 mM Tris–HCl. The total recovery and integrity of these fractions were determined by Lowry et al.46 (link).
5
Brain RNA Extraction and cDNA Synthesis
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For RNA extraction, a subsample of 9 fish per genotype were euthanised with an overdose of tricaine solution (MS222, Pharmaq; 500–1000 mg/L), the whole brain was collected directly into 500 µl lysis buffer (RNeasy Lipid Tissue Mini Kit-Qiagen) and stored at − 80◦C. Total RNA was extracted with the RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer’s instructions, and the concentration, as well as the purity ratios (260 nm/280 nm and 260 nm/230 nm) estimated in the NanoDrop 2000 (Thermo Scientific). The same concentration of RNA for each sample was then reverse transcribed to cDNA (iScript cDNA Synthesis Kit, Biorad), following the manufacturer’s instructions. Briefly, a mix of nuclease-free water, 5 × iScript reaction mix (4 µl), iScript reverse transcriptase (1 µl), and RNA template were prepared in a 1.5 µl sterile tube in a final volume of 20 µl, and incubated in a PCR thermocycler in the following conditions: 5-min priming at 25 °C, 60-min reverse transcription at 42 °C, 5-min reverse transcription inactivation at 85 °C, and then and kept at 4 °C until tube collection. The samples were subsequently stored at -20 °C until further use.
6
RNA Extraction from Lipid Tissue
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The RNeasy Lipid Tissue Mini Kit with QIAzol Lysis Reagent (Quiagen, MD, USA) was used for the extraction according to the manufacturer’s protocol. The obtained RNA was quality controlled on an Agilent 2100 Bioanalyzer system with an RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA). Only those samples with an RNA Integrity Number (RIN) value ≥ 8 were chosen for further analysis.
7
Isolation of Adipose Cell Fractions
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Stromal cells and mature adipocytes were isolated from epididymal adipose tissue from WT and NRF2−/− male mice. Adipose tissue was harvested, weighed, finely minced and digested in HBSS containing 1 mg/mL collagenase type II (Sigma) for approximately 40 min at 37 °C with gentle shaking. The digested material was passed through a 100 μm filter (Falcon-352360), washed with HBSS+10%FBS+P/S and spun at 300 g × 5min. The medium was removed, floating mature adipocytes and stromal vascular fraction (SVF) were collected and washed again. Isolation of mRNA was performed by RNeasy Lipid Tissue Mini Kit (Quiagen). Adiponectin (ADIPOQ), a late marker of adipocyte differentiation, was used to confirm the purity of the fractions.
8
Glioblastoma Multiforme RNA Extraction and Microarray Analysis
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Approximately 50 mg of tissues from the initial 54 GBM samples were used to extract total RNA using the RNeasy Lipid Tissue mini kit (Quiagen, CA), following the instructions of the manufacturer. The quality of RNA obtained was checked using a Bioanalyser System (Agilent Technologies, Paolo Alto, CA) using the RNA Nano Chips. Genome U133 Plus 2.0 Expression arrays data processing was done according to the manufacturer recommendations. Normalization was performed using the RMA method.71 Clustering analysis and class comparison using a univariate t‐test were performed using dChip software (http://biosun1.harvard.edu/complab/dchip/ ).72 A p‐value <.005 was used to define differentially expressed genes. In order to compare the gene expression profile of the gliomas with normal brain, we used the gene expression data of five samples of corpus callosum (GSM175855, GSM175856, GSM175857, GSM175858, GSM176050) and five samples of cortex (GSM176049, GSM176344, GSM176345, GSM176346, GSM176347), available in the Gene Expression Omnibus repository (GSE7307).
9
GLP-1R Expression in Rat Brain Regions
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Brains were collected from 17-week-old rats, frozen, and placed in the cryostat. SuM, LH, nucleus accumbens (encompassing both the shell and the core regions), and NTS were collected using disposable biopsy punches with plungers (INTEGRA, USA) to assess GLP-1R expression using quantitative real-time PCR (qPCR). RNA was isolated using the RNeasy Lipid Tissue Mini Kit (Quiagen, Germany) and GLP-1R levels were quantified using Taqman gene expression kits from Life Technologies using beta actin as the housekeeping gene (Primer information: GLP-1R: Rn00562406_m1; beta actin: RN00667869_m1). Comparative threshold cycle method [31] (link) was used to quantify relative mRNA expression. For both sexes SuM GLP-1R expression relative to beta actin was set as 1 to visualize the expression in SuM relative to other brain regions.
10
Quantification of LHA Gene Expression
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