Protocol for Total RNA Extraction/Purification

Acquisition of high-quality RNA is the first and critical step in performing RNA analysis, including PCR, reverse transcription real-time PCR (RT-qPCR), gene expression profiling with microarray, RNA sequencing, and northern analysis, etc. Total RNA extraction (also known as RNA purification) is the process to extract RNA molecules from biological samples, such as cell culturing, tissue, FFPE, blood. The most important issues involved in the RNA purification process include RNA integrity, purity, and sufficient amount. High-quality RNA is free of genomic DNA or inhibitors, and is not degraded.

Next, we will introduce a manual method of RNA extraction/purification that is suitable for all types of tissues from a majority of plant and animal species. The protocol is slightly variable between scientists. You can also choose commercially available kits that utilize spin column or magnetic beads. RNA is very unstable, so all the reagents and materials should be free of RNase.

Materials:

• Fresh TRIzol Reagent: 40%w/w Phenol, 1M guanidine thiocyanate, 1M Ammonium thiocyanate, 0.1 M sodium acetate buffer (PH=5.0), 5%w/w glycerol
• Chloroform-isoamyl alcohol mix (24:1)
• 100% isopropanol
• 70% ethanol
• 10 M LiCl
• Fresh RNase-free ddH₂O or autoclaved 1xTE

Equipment and consumables

• MM300 Mixer Mill
• Table microcentrifuge
• Vortexer
• Pipettors
• Microcentrifuge tubes

1. Freeze 2ml Eppendorf microcentrifuge tube with tissue sample and glass ball at -80°C and then grind the sample in the MM300 Mixer Mill at 30Hz for two minutes.

2. Add 1 ml fresh TRIzol Reagent to the mechanically disrupted tissue sample, vortex well, and incubate the sample at room temperature for 5 minutes.

3. Add 0.2 ml of chloroform to the microcentrifuge tube for homogenization. Vortex well, and incubate the sample at room temperature for 3 minutes.

4. Centrifugate the sample at maximum speed on table microcentrifuge at 4°C for 5 minutes.

5. Transfer the aqueous phase to a fresh 2 ml microcentrifuge tube. Add an equal volume of chloroform to the fresh microcentrifuge tube, vortex well. Spin at maximum speed on table microcentrifuge for 5 minutes.

6. Transfer the aqueous phase to a fresh 2 ml microcentrifuge tube. Add an equal volume of isopropanol to the fresh microcentrifuge tube, vortex well. Spin at maximum speed on table microcentrifuge at 4°C for 10 minutes.

7. Wash the pellet with 1.5 ml 70% ethanol. Spin at maximum speed on table microcentrifuge for 5 minutes.

8. Dissolve the pellet in 400 μl 1xTE at 55°С well with vortex. Add an equal volume of 10 M LiCl to the solution and chill it at -20°C for several hours (or overnight).

9. Spin the tube at maximum speed on table microcentrifuge at 4°C for 10 minutes. Carefully remove and discard the supernatant (contains DNA and small RNA < 200 nt).

10. Wash pellet with 1.5 ml 70% ethanol, vortex well. Spin the tube at maximum speed on table microcentrifuge at 4°C for 10 minutes. Discard the ethanol, do not dry the pellet.

11. Dissolve the pellet in 200-400 μl fresh RNase-free ddH₂O or 1xTE.

How to check the quality of RNA?

Electrophoresis can be used for the quality check of RNA, including amount and integrity. Load 5 μl of the solution onto a standard 1.5 % agarose gel with 1xTHE buffer. Add ethidium bromide (EB) to the gel. Load a known amount of DNA in a neighboring lane to act as concentration standard. Intact RNA exhibits sharp band(s) of ribosomal RNA.

Additional Readings:

The Methods for DNA Extraction and Purification

Recovering Plasmid DNA from Bacterial Cultures without Using Kits

High-throughput Sequencing Sample Submission Guidelines