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.
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 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.
- 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.
- Remove culture media from flask/plate and wash once with 1x PBS.
- Add miRNA/scramble-transfecting agent complexes and incubate at 37 °C with 5% CO2 for 6 h (flask size defines the added incubation volume).
- Replace the media with 4 mL of culture media and incubate cells for 24 h and/or 48 h.
- 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.
- 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.
- Add 2 mL of 1x PBS and centrifuge for 5 min at 751 x g.
- 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
- Clean the work area by spraying with 70% isopropyl alcohol and RNAse decontamination solution.
- RNA extraction should be performed using an appropriate RNA kit (see Table of Materials).
- 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.
- Add an equal volume of 100% ultra-pure ethanol.
- Mix well and place in separating column.
- Centrifuge between 11 and 16 x g for 30 s and remove the flow-through.
- Add 400 µL of washing buffer and centrifuge to remove the buffer.
- Add 5 µL of DNAse I with 75 µL of DNA digestion buffer in each sample and incubate for 15 min.
- Wash the sample with 400 µL of RNA prep buffer.
- Wash 2x with RNA wash buffer.
- Add nuclease-free water to the column, centrifuge, then collect the RNA.
- Measure total RNA concentration with a UV spectrophotometer.
|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|
|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|