Total RNA from lymphocytes, monocytes, neutrophils and human umbilical vein endothelial cells (HUVECs) was extracted using TRI Reagent (Sigma). Inflammatory genes were evaluated as described elsewhere.10 List of primers used is displayed in Table S2 . Total RNA, including the miRNA fraction, was extracted from serum by using the QIAzol miRNeasy kit (Qiagen), as previously reported.13 The protocols and primers used for both, miRNA profiling and RT‐PCR, are described in the Appendix S1 .
Qiazol mirneasy kit
Manufactured by Qiagen
Sourced in United States
The QIAzol miRNeasy kit is a laboratory product designed for the isolation and purification of total RNA, including small RNA molecules such as microRNA (miRNA), from various biological samples. The kit utilizes a guanidinium thiocyanate-phenol-chloroform extraction method to effectively capture and separate RNA from DNA and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Tri reagent, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Mouse m csf, by BioLegend (1 mentions)
Collagenase d, by Roche (1 mentions)
Dispase, by Worthington (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Human serum plasma mirna pcr array, by Qiagen (1 mentions)
Mircury lna universal rt microrna pcr system, by Qiagen (1 mentions)
6 protocols using qiazol mirneasy kit
1
Extraction and Analysis of Inflammatory Genes and miRNA from Various Cell Types
2
Studying Macrophage Inflammatory Response
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Bone marrow was isolated from WT or miR-146a-/- mice, RBC lysed, and plated in DMEM complete media with 20 ng/mL mouse MCSF (Biolegend). At 4 days of culture, fresh media containing MCSF was added to the cells. At day 7, cells were stimulated with 1 μg/mL LPS (Sigma) for 24 hours. At 24-hours, media was removed and cells were stimulated with a second hit of fresh media containing LPS for an additional 2–6 hours. For Traf6i experiments, cells were pre-incubated with 20 μM Traf6i for 1 hour prior to each LPS stimulation. Protein lysate or RNA was collected using Qiazol/miRNeasy kit (Qiagen) and Western or qRT-PCR was performed on these cells.
3
Adipocyte and Stromal Vascular Fraction Isolation
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Reproductive VAT pads were removed from mice, minced, and digested in buffer containing HBSS, Collagenase D (Roche), and Dispase (Worthington) for 1 hour at 37 degrees. Homogenates were placed on ice for 30 minutes then spun to separate adipocytes from SVF. Adipocytes, which float on the top, were considered the adipocyte fraction. Supernatant was then removed to obtain the pelleted SVF. RBC lysis buffer was added to the SVF, which was spun and washed. RNA was collected from the adipocyte and SVF fractions via Qiazol/miRNeasy Kit (Qiagen), and miR-146a levels were measured using miRCURY LNA RT PCR (Exiqon) and the mmu-miR-146a primer from Exiqon. Primer sequences for CD45 and Leptin can be found in supplemental materials and methods.
4
Serum miRNA Extraction Protocol
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Total RNA, including the miRNA fraction, was extracted from serum by using the QIAzol miRNeasy kit (Qiagen, Valencia, CA, USA) with some modifications. A total of 200 μl of serum were thawed on ice and lysed in 1 mL QIAzol Lysis Reagent (Qiagen). Samples in QIAzol were incubated at room temperature for 5 min to inactivate RNases. To adjust for variations in RNA extraction and/or copurification of inhibitors, 5 fmol of spike-in non-human synthetic miRNA (C. elegans miR-39 miRNA mimic: 5′-UCACCGGGUGUAAAUCAGCUUG-3′) were added to the samples after the initial denaturation. The remaining extraction protocol was performed according to the manufacturer’s instruction. Total RNA was eluted in 14 μl of RNase-free water and stored at −80°C.
5
Extracting and Analyzing miRNA from Plasma
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Total RNA, including the miRNA fraction, was extracted from both plasma and supernatants obtained from in vitro studies by using the QIAzol miRNeasy kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions13 (link) (Online Supplementary Appendix). To identify the changes that occurred in the expression levels of miRNAs in plasma from APS patients and HDs, a Human Serum & Plasma miRNA PCR-array (Qiagen) was performed (Online Supplementary Appendix) on an exploratory cohort (Online Supplementary Table S2).
6
Plasma miRNA-221 and miRNA-222 Levels
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In rats, miRNA-221 and miRNA-222 were measured in plasma from five animals of the vehicle group and six from FGF23-treated animals.
In patients, the amount of miRNA-221 and miRNA-222 was measured in seven patients with the highest serum concentration of C-FGF23 (highest decile) and seven patients with the lowest levels of C-FGF23 (lowest decile). Total RNA, including the miRNA fraction, was isolated from a plasma sample of 200 µl using the QIAzol miRNeasy kit (Qiagen) according to the manufacturer’s instructions. Due to the absence of an endogenous miRNA control in plasma, a non-human synthetic miRNA was added to each of the samples as an internal control (C. elegans miR-39 mimic: 5′-UCACCGGGUGUAAAUCAGCUUG-3′; Qiagen). Finally, the total RNA was eluted in 14 µl of RNAse-free water. cDNA was synthesized from 3 µL of each sample using miRCURY LNA Universal RT microRNA PCR system (Qiagen) according to manufacturer's instructions. The 2−ΔCt method was used to calculate the relative abundance of miRNA compared with miR-39 as an internal control.
In patients, the amount of miRNA-221 and miRNA-222 was measured in seven patients with the highest serum concentration of C-FGF23 (highest decile) and seven patients with the lowest levels of C-FGF23 (lowest decile). Total RNA, including the miRNA fraction, was isolated from a plasma sample of 200 µl using the QIAzol miRNeasy kit (Qiagen) according to the manufacturer’s instructions. Due to the absence of an endogenous miRNA control in plasma, a non-human synthetic miRNA was added to each of the samples as an internal control (C. elegans miR-39 mimic: 5′-UCACCGGGUGUAAAUCAGCUUG-3′; Qiagen). Finally, the total RNA was eluted in 14 µl of RNAse-free water. cDNA was synthesized from 3 µL of each sample using miRCURY LNA Universal RT microRNA PCR system (Qiagen) according to manufacturer's instructions. The 2−ΔCt method was used to calculate the relative abundance of miRNA compared with miR-39 as an internal control.
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