Cells and tissues were homogenized and lysed using TRIzol Reagent (Invitrogen) and RNA was extracted using RNeasy Plus Mini kit columns (Qiagen, Hilden, Germany). The cDNAs were synthesized using the GoScriptTM reverse transcriptase kit (Promega, Madison) and assayed using the SYBR® Premix Ex Taq™ II (Takara, Shiga, Japan) and gene‐specific primers listed in supplementary information Table 1 and our earlier study.44 (link) Gene expression was determined by the 2−ΔΔCt method with amplification efficiencies ranging between 90 and 110% for the target and reference genes.
Rneasy plus mini kit columns
Manufactured by Qiagen
Sourced in Germany, United States
The RNeasy Plus Mini kit columns are used for the purification of total RNA from a variety of sample types. The columns provide efficient and reliable RNA extraction, enabling the isolation of high-quality RNA for subsequent analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Sybr premix ex taq 2, by Takara Bio (3 mentions)
Goscript reverse transcriptase kit, by Promega (2 mentions)
Tripure reagent, by Roche (2 mentions)
Chloroform, by Fujifilm (1 mentions)
Trypsin solution, by Fujifilm (1 mentions)
Agilent 2200 tapestation, by Agilent Technologies (1 mentions)
Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
5 protocols using rneasy plus mini kit columns
1
Gene Expression Analysis by qRT-PCR
2
RNA Extraction and qRT-PCR Analysis
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Cells and tissues were lysed using TRIzol Reagent (Invitrogen) for RNA extraction and cDNAs synthesis using RNeasy Plus Mini kit columns (Qiagen, Hilden, Germany) and GoScriptTM reverse transcriptase kit (Promega, Madison, USA), respectively. SYBR® Premix Ex Taq™ II (Takara, Shiga, Japan) was used for performing qRT-PCR with gene-specific primers described in Supplementary Table 2 . Gene expression was calculated by the 2−ΔΔCt method after normalizing to the GAPDH and β-actin37 (link).
3
RNA Extraction and qRT-PCR Analysis
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Cells and homogenized tissues were lysed using TRIzol Reagent (Invitrogen) for RNA extraction. To obtain RNA from calvarial bones following implantation of wear particles, frozen samples were crushed using tissue homogenizer (Power Masher II, Nippi, Tokyo, Japan) in liquid nitrogen. Cells (1 × 106) from the granulomatous tissue around UHMWPE particles were obtained on day 7 and then digested by trypsin solution (Wako) for 10 min in 37°C-water bath. Thereafter, chloroform (Wako) was added for phase separation and the DNA-free RNA was purified from the aqueous layer using RNeasy Plus Mini kit columns (Qiagen, Hilden, Germany). Purified RNA (0.5 μg) was reverse transcribed using the GoScriptTM reverse transcriptase kit (Promega, Madison, USA) in order to carry out a qRT-PCR analysis. The cDNAs were assayed using the SYBR® Premix Ex Taq™ II (Takara, Shiga, Japan) and gene-specific primers listed in Supplementary Table (13 (link)). Specific primers were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) to amplify 60–100 bp of the genes. The gene expression was calculated by the 2−ΔΔCt method after normalizing to the expression of the housekeeping genes, including GAPDH and β-actin. Amplification efficiencies of the target and reference genes ranged between 90 and 110%.
4
Spleen Tissue Sampling and RNA Isolation
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Animals selected for this study were comingled in a pen with ad libitum access to the same diet. Animals were slaughtered over a consecutive 4-day period, with one animal from each of the phenotypic groups represented during each harvest day. The steers were able to consume feed and water until their weights were taken on the morning of slaughter and transported to the US Meat Animal Center abattoir (under 6.4 km). On each slaughter day, the four animals selected were processed serially within a 3-h time frame. The spleen tissue was obtained from each steer at ~30 min post-exsanguination. A portion of the spleen was removed from the unattached end of the spleen, ~8–10 cm from the free end of the spleen. Small sections of the spleen tissue were diced into 50–100 mg samples from middle of the open end of the spleen slice. These samples were immediately frozen in liquid nitrogen and stored at −80°C until they could be processed further. RNA was isolated with TriPure Reagent (Roche, Indianapolis, IN, USA) according to the manufacturer's protocol with an extra 20 min centrifugation following the addition of chloroform. The resulting RNA pellets were centrifuged through RNeasy Plus Mini Kit columns (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions to remove genomic DNA.
5
Mesenteric Fat RNA Extraction and Analysis
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On each day of slaughter, the four animals selected were processed serially within a three-hour time frame. The tissue sample was obtained from each steer within approximately 30–40 minutes after exsanguination. Mesenteric fat was removed from approximately 1-m distal to the duodenum. Mesenteric fat samples were diced into 50–100 mg pieces and flash frozen in liquid nitrogen then stored at -80°C until they could be processed further. Tissue was homogenized in TriPure reagent (Cat # 11667165001; Roche, Indianapolis, IN, USA). RNA was isolated according to the manufacturer’s protocol with an extra 20-minute centrifugation following the addition of chloroform. The resulting RNA pellets were further processed using RNeasy Plus Mini Kit columns (Cat # 74136; Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions to remove genomic DNA.
The concentration of the RNA was determined with a Nanodrop 8000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The 260/280 ratios were ≥ 1.8 and a subset of 20 samples (at least 3 samples from each cohort) were analyzed for quality on either the Agilent Bioanalyzer 2100 or the Agilent Tapestation 2200 (Cat #5067-1511 and 5067–5576; Agilent Technologies, Santa Clara, CA, USA) and produced an average RIN of 8.2 with a range of 7.1 to 9.2.
The concentration of the RNA was determined with a Nanodrop 8000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The 260/280 ratios were ≥ 1.8 and a subset of 20 samples (at least 3 samples from each cohort) were analyzed for quality on either the Agilent Bioanalyzer 2100 or the Agilent Tapestation 2200 (Cat #
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