RNA Extraction

Overview:
Total RNA is isolated and separated from DNA and protein after extraction
with a solution called as Trizol. Trizol is an acidic solution containing
guanidinium thiocyanate (GITC), phenol and chloroform. GITC irreversibly
denatures the DNA which is a process in which proteins or nucleic acids
lose the quaternary structure, tertiary structure and secondary structure
which is present in their native state.
This is followed by centrifugation. Under acidic conditions, total RNA
remains in the upper aqueous phase, while most of DNA and proteins
remain either in the interphase or in the lower organic phase.
Total RNA is then recovered by precipitation with isopropanol.

Learning objectives:
1. To learn how to extract Total RNA from the tissue.
2. Understand how to protect RNA samples.

Materials and Equipment:

- Trizol reagent.
- Centrifuge.
- Tissue sample.
- Chloroform.
- Water bath.
- Isopropanol.
- Ethanol 70%.
- RNase free water (DEPC H2O).
- Ethanol atomizer 70%.
- Hydrogen Peroxide atomizer 3%.
- Homogenizer.
- TE buffer.
- Eppendorf.
Procedure:
Stage one: Homogenization
1. Clean your working area with ethanol 70% and 3%hydrogen peroxide by
atomizer.

2. Add 1 mL TRIZOL reagent to the tissue then homogenize the sample

using homogenizer.

3. Incubate the sample for 5 minutes at room temperature to allow

complete dissociation.

4. Transfer the tissue homogenate to a new eppendorf.

Stage two: Phase separation

5. Add 200 μL of chloroform to the sample then close the cap of the tube
then make inversion twice.

6. Incubate the sample for 3 minutes at room temperature.

7. Place the sample in the centrifuge for 15 minutes at 12000 rpm and 4 °C.

Note: by the end of the centrifugation you will be able to see upper
colorless aqueous phase and interphase and lower red organic phase.

8. Transfer the aqueous layer carefully to a new tube.

Note:
- Avoid transferring any of the interphase or organic lower layer into the
pipette while transferring the aqueous phase.
- Store the interphase and organic phase at 4 °C in order to use them to
isolate DNA or Protein.

Stage three: RNA precipitation

9. Add 500 μL of Isopropanol to the aqueous phase then close the cap of
the tube then make inversion twice.

10. Incubate the sample for 10 minutes at room temperature.

11. Place the sample in the centrifuge for 15 minutes at 12000 rpm and 4 °C.

Note: by the end of centrifugation you will be able to see total RNA
precipitate as a white pellet at the bottom of the tube.

Stage four: RNA washing

12. Remove the supernatant using pipette.

13. Wash the pellet with 1 mL of 70 % ethanol.

14. Close the cap of the tube then make inversion twice.

15. Place the sample in the centrifuge for 5 minutes at 7500 rpm and 4 °C.

16. The ethanol supernatant is removed using micropipette.

17. Leave the RNA to dry in air for 5 minutes.

18. Suspend the pellet in TE buffer by adding 50 μL of RNase free TE buffer

then make pipetting.

19. Incubate the pellet in water bath at 60 4 °C for 15 minutes.

Now you have RNA sample ready for use in any other processes.