RNA Extraction Assay: A Method to Extract RNA from miRNA Transfected Lung Cancer Cells
Published: April 30, 2023
Abstract
Source: Hossian, A. K. M. N. et al., Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis. J. Vis. Exp. (2019).
This video describes the RNA extraction protocol from miRNA transfected lung cancer cells. This approach can be used to identify a combinatorial miRNA treatment activity in halting the cell cycle and angiogenesis.
Protocol
1. miR-143 and miR-506 transfection CAUTION: Use latex gloves, protective eyeglasses, and a laboratory coat while performing the described experiments. When required, use the biosafety cabinet with the cabinet fan on, without blocking the airways or disturbing the laminar airflow. Always set the protecting glass window to the appropriate height, as described by the manufacturer. Seed NSCLC A549 cells in a T25 cm2 flask/6/96 well plate in…
開示
The authors have nothing to disclose.
Materials
| A549 Non Small Cell Lung Cancer Cells | ATCC | ATCC CCL-185 | |
| Ambion Silencer Negative Control No. 1 siRNA | Ambion | AM4611 | |
| hsa-miR-143-3p miRNA Mimic | ABM | MCH01315 | |
| hsa-miR-506-3p miRNA Mimic | ABM | MCH02824 | |
| Fetal Bovine Serum (FBS) | Fisher Scientific | 10438026 | |
| HyClone Phosphate Buffered Saline (PBS) | Fisher Scientific | SH30256FS | |
| DMEM 4.5 g/L Glucose, w/out Sodium Pyruvate, w/ L-Glutamine | VWR | VWRL0100-0500 | |
| Invitrogen UltraPure DNase/ RNase-Free Distilled Water | Invitrogen | 10-977-015 | |
| Quick-RNA Kits | Zymo Research | R1055 | |
| Ethanol, Absolute (200 Proof), Molecular Biology Grade, | Fisher BioReagents | BP2818500 | |
| -80 °C Freezer | VWR | VWR40086A | |
| Nanodrop | Thermo Scientific | NanoDrop one C | |
| 96 well plate | CELLTREAT Scientific | 50-607-511 | |
| Penicillin-streptomycin 10/10 | Antlanta Biologicals | B21210 | |
| CO2 Incubator | Thermo Scientific | HERAcell 150i | |
| dnase | Zymo Research | E1010 | |
| F-12K Nutrient Mixture (Kaighn's Mod.) with L-glutamine, Corning | Corning | 45000-354 | |
| Fetal Bovine Serum – Premium | Antlanta Biologicals | S11150 | |
| Temperature controlled centrifuge matchine | Thermo Scientific | ST16R | |
| Temperature controlled micro centrifuge matchine | Eppendrof | 5415R |
