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

- 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, se