- To begin, seed the lung cancer cells in a T-flask containing tissue culture medium and let it grow overnight. Next, suspend miRNA with a transfecting agent into a transfecting medium. Remove media and add a transfecting medium that will introduce miRNA into cancer cells. miRNA is a non coding RNA molecule that binds to a complementary mRNA sequence and blocks gene expression. Incubate the flask for the desired duration.
Next, replace the media with tissue culture media and grow overnight. Add trypsin for cell dissociation. Transfer the mixture to a centrifuge tube containing PBS and centrifuge it. Remove the supernatant and add lysis buffer to lyse cells and add ultra pure ethanol for precipitation. Place the mixture in a separating column to purify nucleic acid and centrifuge.
Remove the flow through and add a wash buffer to remove the lysed cell membrane and centrifuge. Remove the wash buffer and add DNase with DNase digestion buffer to remove DNA from the sample and centrifuge. Add RNA prep buffer to isolate RNA and centrifuge. Collect the RNA present in the supernatant. In the following protocol, we will perform RNA extraction from miRNA treated lung cancer cells.
- First, seed the cancer cells in an appropriate flask sufficient for RNA and protein extraction to perform gene expression using qPCR or microarray analysis. Then incubate overnight with media supplemented with 10% FBS and 1% penicillin-streptomycin.
The next day prepare the microRNA sample for transfection by diluting microRNA 143 and microRNA 506 in the media, at a concentration of 100 nanomolar each. Take out the flask from the incubator, and remove the media. Wash with 1 x PBS.
All the untreated controls will contain only incomplete media with cells. Incubate the cells with transfection media for six hours in an incubator. After six hours, remove the media and replace with 4 mililiters of fresh complete media. Harvest the cells after 24 hours and 48 hours by trypsinization.
Next wash twice with 1x PBS in each tube, and remove supernatant. In order to perform RNA extraction at a later time, freeze the cells at minus 80 degrees Celsius.
- RNA extraction was performed using an RNA kit, following manufacturer's instructions. First remove the tubes from minus 80 degrees Celsius and allow to thaw.
- Add 300 microliters of lysis buffer and pipette up and down to break the cell membrane. Add equal volume of 100% ultra pure ethanol. Then mix well and place in the separating column. Centrifuge and remove the flow through.
Add 400 microliters of washing buffer and centrifuge to remove the buffer. Add 5 microliters of DNAse I with 75 microliters of DNA digestion buffer in each sample and incubate for 15 minutes. Then wash the sample with 400 microliter RNA prep buffer.
Next wash twice with RNA wash buffer. Now add nuclease-free water to the column, centrifuge and then collect the RNA. Measure the RNA concentration, in this stage, 2 micrograms of RNA sample can be sent for the RNA sequencing.