Total RNA & Protein Extraction from Peripheral Blood Mononuclear Cells

Protocol Overview

Cells were pelleted via centrifugation. The pellet was washed with 1 mL PBS, re-pelleted as above and then stored on ice. Cells were lysed by addition of ice-cold Cell Disruption Buffer and vortexing. Lysate was stored on ice and split into two, one-half for total RNA extraction and the other for protein isolation. Part of the lysate that was used for the protein extraction which was incubated on ice, then passed through a syringe needle several times on ice for further homogenisation. Extracted protein was then stored at – 20 ºC until further use. The lysate that was used for RNA isolation was mixed with an equal volume of 2X Denaturing Solution at room temperature. A volume of Acid-Phenol: Chloroform that was equal to the total volume of the sample lysate and 2X Denaturing Solution was added to the mixture and vortexed. The mixture was centrifuged. Carefully, the upper aqueous phase was removed and transferred it to a fresh tube. 100% ethanol was added to the aqueous phase and loaded onto a Filter Cartridge and centrifuged. Total RNA was washed by addition of Wash Solution 1, centrifuged and then Wash Solution 2/3 (500 μL) was as mentioned above. This step was repeated again.Total RNA was eluted using nuclease-free water preheated at 95 degrees C. The concentration and purity of total RNA was determined using the Nanodrop.