RNA Extraction Protocol in Detail

RNA Extraction Protocol in Detail

Let’s dive into details using a widely accepted protocol based on an RNA extraction kit (which utilizes the organic extraction method with TRIzol, which is a monophasic solution of phenol and guanidine isothiocyanate). For this example, I will use the one made by Thermo Fisher Scientific.

Materials

1. TRIzol Reagent

2. Chloroform

3. Isopropanol

4. 75% Ethanol

5. RNase-free water

6. RNase-free gloves, tubes, pipette tips, and all other equipment

7. Stored sample (either tissue or cells are fine)

8. Microcentrifuge

9. Micropipettes

10. Vortex

Procedure

Please remember to wear your lab coat and safety goggles before starting the process.

1. Homogenization

   – Add 1 ml of TRIzol Reagent to every 50–100 mg of tissue or 5 × 106 cells. Homogenize the tissue or resuspend the cells in the reagent thoroughly. This step is performed to lyse the cells and denature cellular proteins.

2. Phase Separation

   – After homogenizing, incubate the samples for 5 minutes at room temperature to permit the complete disassociation of nucleoprotein complexes.

   – Now, add 200 μl of chloroform per 1 ml of TRIzol Reagent. Shake the tubes vigorously by hand for 15 seconds and incubate them at room temperature for 2 to 3 minutes.

   – Centrifuge the samples at 12,000 × g for 15 minutes at 4°C.

3. RNA Precipitation

   – Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains in the aqueous phase.

   – Transfer the aqueous phase to a fresh tube.

   – Precipitate the RNA from the aqueous phase by mixing it with isopropanol. Use 500 μl of isopropanol per 1 ml of TRIzol Reagent used for the initial homogenization.

   – Incubate samples at room temperature for 10 minutes and centrifuge at 12,000 × g for 10 minutes at 4°C.

4. RNA Wash

   – Remove the supernatant and wash the RNA pellet once with 1 ml of 75% ethanol per 1 ml of TRIzol Reagent used for the initial homogenization.

   – Mix the sample by vortexing and then centrifuge at 7,500 × g for 5 minutes at 4°C.

5. RNA Hydration

   – At the end of the procedure, remove the supernatant and air-dry the RNA pellet for about 5–10 minutes.

   –  Dissolve RNA in RNase-free water by passing the solution a few times through a pipette tip, and incubating for 10 minutes at 55 to 60°C.

6. RNA Quantification

   – Quantify the RNA by measuring the absorbance at 260 nm in a spectrophotometer.  

Remember to maintain RNase-free conditions during the procedure to prevent RNA degradation. This includes wearing gloves, using RNase-free tubes and solutions, and, whenever possible, working in an RNase-free environment. Always follow the specific protocol related to the kit you are using, as various manufacturers may have unique steps for their products.

Please note, that this protocol is intended to be a guideline. The success of RNA extraction can depend on many factors, including the age and type of the sample and the exact method of extraction. Always consult with a lab supervisor or experienced peers if you aren’t sure about any step.

Dr AF Saeed

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