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

Samenvatting

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 °C with 5% CO₂ in a tissue culture incubator.
2. Suspend miR-143 and/or miR-506 mimics, or scramble siRNA with transfecting agent (2.4 µg of miR were mixed with 14 µL of transfecting agent; see Table of Materials) in 500 µL of transfection media and 1.5 mL of serum and antibiotic-free DMEM/F12K media at a final miRNA concentration of 100 nM. miRNA amount may require optimization on different cells and concentrations. Appropriate approaches include the transfection of cells with increasing concentrations of miRNA (i.e., 50-200 nM) and evaluation of expression downregulation of the genes of interest.
3. Remove culture media from flask/plate and wash once with 1x PBS.
4. Add miRNA/scramble-transfecting agent complexes and incubate at 37 °C with 5% CO₂ for 6 h (flask size defines the added incubation volume).
5. Replace the media with 4 mL of culture media and incubate cells for 24 h and/or 48 h.
6. Harvest transfected cells by trypsinization, by adding 1 mL of trypsin-EDTA in each T25 cm² flask, incubate for 5-10 min at 37 °C, and add 3 mL of culture media to harvest the cells. Place the contents of each flask into a separate, marked 15 mL tube. Work in a tissue culture hood.
7. Centrifuge at 751 x g for 5 min and remove the supernatant.
   NOTE: Caution is required during supernatant removal, as agitation of the tube may cause loss of cells.
8. Add 2 mL of 1x PBS and centrifuge for 5 min at 751 x g.
9. Repeat steps 7 and 8 once to remove any traces of media and supernatant.
   NOTE: At this stage, sample tubes can be stored at -80 °C or can be used immediately.

2. RNA extraction

1. Clean the work area by spraying with 70% isopropyl alcohol and RNAse decontamination solution.
2. RNA extraction should be performed using an appropriate RNA kit (see Table of Materials).
3. Remove the tubes from -80 °C and allow them to thaw. Add 300 µL of lysis buffer and pipette up and down to break the cell membrane.
4. Add an equal volume of 100% ultra-pure ethanol.
5. Mix well and place in separating column.
6. Centrifuge between 11 and 16 x g for 30 s and remove the flow-through.
7. Add 400 µL of washing buffer and centrifuge to remove the buffer.
8. Add 5 µL of DNAse I with 75 µL of DNA digestion buffer in each sample and incubate for 15 min.
9. Wash the sample with 400 µL of RNA prep buffer.
10. Wash 2x with RNA wash buffer.
11. Add nuclease-free water to the column, centrifuge, then collect the RNA.
12. Measure total RNA concentration with a UV spectrophotometer.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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