ECM isolation was modified from published protocols for adipose and lung tissues (8–10 (link)). VAT explants (1 g) were freeze-thawed in 10 mM Tris, 5 mM EDTA, and 1% phenylmethanesulphonylfluoride (PMSF) (pH 8.0) from −80°C, 20 minutes to 37°C, 5 minutes 3 times. Explants were incubated at 37°C for 24 hours in 0.25% Trypsin/0.1% EDTA; washed in rinsing buffer (8 g/L NaCl, 200 mg/L KCl, 1 g/L Na2HPO4, 200 mg/L KH2PO4, 1% PMSF) at 37°C for 20 minutes three times; incubated at 37°C for 24 hours in 55 mM Na2HPO4, 17 mM KH2PO4, 4.9 mM MgSO4⋅7H2O, 160 U/mL DNase I type II, 100 μg/mL RNase type IIIA, 80 U/mL lipase type VI-S (Sigma-Aldrich, Inc., St. Louis MO, USA), and 1% PMSF; and washed sequentially in rinsing buffer at 37°C for 20 minutes three times; once in 99.9% isopropanol, 1% PMSF at 25°C for 24 hours; rinsing buffer at 37°C for 20 minutes three times; 70% EtOH at 37°C for 20 minutes three times; and storage solution (PBS, 1% PMSF) at 37°C for 20 minutes once. Explants were stored in storage solution 4°C.
Rnase type iiia
Manufactured by Merck Group
Sourced in United States, Italy
RNase type IIIA is a laboratory enzyme that degrades RNA molecules. It functions by cleaving single-stranded RNA, particularly small interfering RNA (siRNA) and microRNA (miRNA) molecules. This enzyme is commonly used in molecular biology research and the development of RNA-based therapeutics.
Automatically generated - may contain errors
2 protocols using rnase type iiia
1
Isolated Extracellular Matrix Extraction
2
Cell Cycle Analysis of Prostate Cancer
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LNCaP, 22Rv1 (5 × 105), PC3 and DU145 (3 × 105) cells were seeded in 60-mm plates and 24 h later they were treated with 0.1% DMSO dissolved in culture medium or with melatonin (10−3 M) or with UCM 1037 (10−4 M) diluted in 0.1% DMSO. After 24, 48, and 72 h cells were collected, washed, and stained for 30 min at 37 °C with 1 mL of DNA-staining solution in 0.1% Nonidet P-40 (Sigma-Aldrich, Milan, Italy), 0.5 mg/mL RNAse (type IIIA, Sigma-Aldrich), and 25 µg/mL propidium iodide (Sigma-Aldrich) [44 (link)]. The cellular DNA content was analyzed by FACScalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) using a Cell Quest (Becton Dickinson) software system for histograms of propidium iodide fluorescence intensity vs. cell frequency.
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