RNA Extraction Assay: A Method to Extract RNA from miRNA Transfected Lung Cancer Cells

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.

1. Seed NSCLC A549 cells in a T25 cm² flask/6/96 well plate in DMEM/F12K media supplemented with 10% FBS and 1% penicillin-streptomycin (culture media) in a tissue culture hood and incubate overnight at 37 °....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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