- After collection and centrifugation, plasma or serum can be stored at 2–8°C for up to 6 hours or used directly in the procedure. For long-term storage, freezing at –20°C or –80°C in aliquots is recommended. Avoid prolonged incubation, which may compromise RNA integrity.
- Except for phase separation (step 7), all protocol and centrifugation steps should be performed at room temperature.
- The procedure is suitable for use with serum samples or with plasma samples containing either citrate or EDTA. Plasma samples containing heparin should not be used because this anticoagulant can interfere with downstream assays, such as RTPCR.
- Buffers RWT and RPE are supplied as concentrates. Before using for the first time, add the required volumes of ethanol (96–100%), as indicated on the bottle, to obtain a working solution.
- The Syn-cel-miR-39 miScript miRNA Mimic (cat. no. MSY0000010) can be added to samples to control for variations during the preparation of total RNA and subsequent steps, as it shows no sequence homology to any known human, mouse, or rat miRNA. miScript miRNA Mimics are provided lyophilized. Prepare the miScript miRNA Mimic stock and working solution as follows:
Stock - Add 300µl RNase-free water to lyophilized miScript Serum/Plasma Spike-In Control (10pmol). Final concentration will be 2 x 1010 copies/µl.
Dilution - Add 4µl stock (2 x 1010 copies/µl) to 16µl RNase-free water. Final concentration will be 4 x 109 copies/µl.
Working solution for RNA purification - Add 2µl of 4 x 109 copies/µl dilution to 48µl RNase-free water. Final concentration will be 1.6 x 108 copies/µl.