Solid material was removed from the pasteurized samples by filtration through a 0.22 µm filter (MilliporeSigma SCGP00525). To precipitate viral particles, 35 mL of filtered wastewater was combined with 5 mL of 80% PEG-8000 and 0.3 M NaCl solution in a 50-mL OakRidge centrifuge tube, vortexed to mix thoroughly, and held on ice until centrifugation. Viral particles were pelleted by centrifugation at 12,000×g for 1.5 h, at 4 °C. To extract viral RNA, virus pellets were resuspended in Qiazol Lysis Reagent (Qiagen 79306) and incubated 5 min at room temperature. Aqueous and organic phases were separated by addition of chloroform, vortexing, and centrifugation at 12,000×g for 15 min at 4 °C. The aqueous phase was transferred to Qiagen miRNeasy mini spin columns (Qiagen 217004) for RNA purification using manufacturer protocol specifications for micro-RNA isolation. Purified RNA was stored at − 80 °C.
Mirneasy mini spin columns
Manufactured by Qiagen
Sourced in Germany
The MiRNeasy mini spin columns are designed for the purification of total RNA, including small RNAs such as miRNA, from a variety of sample types. The columns use a silica-based membrane to selectively bind RNA molecules, allowing for the efficient capture and purification of RNA from the sample.
Automatically generated - may contain errors
Lab products found in correlation
Scgp00525, by Merck Group (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
Qiazol reagent, by Qiagen (2 mentions)
Hiflex buffer, by Qiagen (1 mentions)
Epoch spectrophotometer, by Agilent Technologies (1 mentions)
Icycler real time pcr detection system, by Bio-Rad (1 mentions)
Miscript reverse transcriptase mix, by Qiagen (1 mentions)
Bead beater 16, by Biospec (1 mentions)
On column dnase digestion, by Qiagen (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
5 protocols using mirneasy mini spin columns
1
SARS-CoV-2 Wastewater Viral Concentration
2
Filtration and Viral RNA Extraction from Wastewater
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Solid material was removed from the pasteurized WW samples by filtration through a 0.02μm filter (MilliporeSigma SCGP00525). To precipitate viral particles, 35 mL of filtered WW was combined with 5 mL of 80% PEG-8000, 0.3M NaCl solution in a 50-mL OakRidge centrifuge tube, vortexed to mix thoroughly, and held on ice until centrifugation. Viral particles were pelleted by centrifugation at 12,000 × g for 1.5 h at 4 C. To extract viral RNA (vRNA), virus pellets were resuspended in Qiazol Lysis Reagent (Qiagen 79306) and incubated 5 min at room temperature. Aqueous and organic phases were separated by addition of chloroform, vortexing and centrifugation at 12,000 × g for 15 min at 4 C. The aqueous phase was transferred to Qiagen miRNeasy mini spin columns (Qiagen 217004) for RNA purification using manufacturer protocol specifications for micro-RNA isolation. Purified RNA was stored at −80 C.
3
Serum miRNA Profiling in HCC and HCV Patients
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This study was approved by the institutional review committee board of Ethics, Ain Shams University, Faculty of Medicine, Egypt, under the Code of Ethics of the World Medical Association (Helsinki Declaration) with the approval number FMASU MD 32/2016. Informed written consent was obtained from every participant. Participants were recruited from the clinic of Tropical Medicine Department, Ain Shams University Hospital in the period from January to April 2016. All patients who had received radiation, chemotherapy or surgical intervention were excluded from the study. Demographic data of the population of patients in this study are summarized in Table 1 . Serum samples from 25 histologically confirmed HCC patients, 15 hepatitis C virus (HCV) patients, and 10 healthy normal participants were used for total RNA isolation using miRNeasy Mini Spin Columns (Qiagen, Hilden, Germany).
4
Quantifying Cardiac miR-21 Expression
5
PMNL Total RNA Extraction
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After incubation, PMNL were pelleted by centrifugation and the supernatant collected for further analysis. Total RNA was extracted using Qiazol reagent (Qiagen, Hilden Germany). The cell pellet was placed in 1 mL of Qiazol and tissue was homogenized with a Bead Beater 16 (Biospec, Bartlesville, OK), using two 30-s cycles of the homogenizer at full speed, and placed on ice for 1 min after homogenization. Homogenized samples were centrifuged to remove any remaining cell debris. Chloroform was then added to the homogenized sample, centrifuged at 16,000 × g for 15 min at 4°C, and the aqueous phase removed carefully. Precipitation of RNA was achieved with the addition of ethanol (Decon Labs Inc., King of Prussia, PA), and the subsequent RNA pellet was washed and cleaned using miRNeasy mini spin columns (Qiagen). Genomic DNA was removed during purification with on-column DNase digestion (Qiagen). The RNA concentration was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and RNA quality was assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). All samples had an RNA integrity value >8.0.
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