Overview:
♦ Tissues are composed of multiple cell layers that are tightly glued together by extracellular matrix.
♦ Solubilizing all cells of tissue in Trizol is tricky and requires extensive homogenization.
♦ Once all cells have been lysed and solubilized, the later steps (phase separation, RNA precipitation etc) are essentially the same as isolating RNA from cell culture.
♦ To minimize the RNA degradation, tissue is snap-frozen in liquid nitrogen as soon as the tissue is dissected out from the animal and then stored at -80°C or in liquid nitrogen until the RNA isolation begins.
♦ The protocol described here is suitable for the isolation of total RNA using Trizol from most tissues.
♦ The isolation procedure is simple, reliable, and reproducible, and yields high-quality RNA. However, traces of genomic DNA can be present in the purified RNA. Therefore, DNase I digestion to remove genomic DNA contamination is recommended if the RNA is to be used for applications that can not afford traces of genomic DNA contamination such as PCR-based expression analysis of genes.
Requirements:
Reagents and solutions:
Trizol
Chloroform
Nuclease-free water
Isopropyl alcohol
75% Ethanol
Equipment and disposables
Micropipette
Micropipette tips
Refrigerated centrifuge
1.5 or 2 ml microcentrifuge tubes
Fume hood
Vortex mixer
Homogenizer
Starting material:
50 – 100 mg snap-frozen tissue
Prior to start
♦ Set the temperature of the centrifuge to 4°C.
Precautions:
♦ Trizol contains GITC and Phenol. Use appropriate safety measures as per your institute’s guidelines and instructions in the supplier’s manual.
♦ All operations related to Trizol must be done inside the fume hood.
♦ Use all reagents and disposables free of RNases.
Procedure
Step 1: Solubilize the tissue in Trizol using homogenizer.
◊ Homogenize and solubilize 50 – 100 mg snap-frozen tissue in 1 ml Trizol.
◊ Incubate at room temperature for 5 min.
◊ If there are insoluble material, centrifuge the lysate to remove it (12,000 g, for 5 min at 4°C).
◊ Transfer lysate to a 1.5 ml microcentrifuge tube using a P1000 micropipette.
Tips:
◊ If tissue is RNase-rich, you can thaw the tissue in RNAlater, and then homogenize the tissue using Dounce homogenizer or any other similar homogenizer.
◊ If tissue is fibrous, tissue must be ground while frozen using Mortar and pestle.
◊ Freshly dissected tissues can directly be collected in RNAlater and can be homogenized immediately for RNA isolation.
◊ Use 1 ml Trizol per 50 – 100 mg tissue. Scale up the Trizol volume accordingly if you have more than 100 mg tissue.
Pause point: Lysate can be stored overnight at 4°C or for up to a year at –20°C.
Step 2: Separate the aqueous and organic phases by centrifugation and transfer the upper aqueous phase to a fresh microcentrifuge tube.
◊ Add 200 µL chloroform and mix by vigorous shaking. Let the centrifuge tube stand on the bench in a straight vertical position for 2–3 min. You will see the upper bit hazy aqueous phase and lower pink organic phase.
◊ Centrifuge the sample at high speed (12,000 × g) at 4°C for 15 min.
◊ Following centrifugation, the upper aqueous phase will become clear and transparent. Between lower pink organic phase and upper clear aqueous phase, you will also see precipitated material (also referred to as interphase).
◊ Transfer the upper clear aqueous phase to a fresh 1.5 ml centrifuge tube.
Tips:
◊ Save the precipitate (the interphase) and organic phase if you want to isolate DNA and protein, otherwise discard the tube.
◊ You can vortex the sample for a few seconds to mix cell lysate and chloroform.
◊ Centrifugation can also be performed at room temperature. Keeping the temperature low helps in the formation of relatively sharp boundaries between aqueous and organic phases.
Note:
The upper aqueous phase contains RNA, the interphase (precipitate) contains precipitated protein and genomic DNA and the lower phase contains soluble proteins and other cellular components.
Precaution:
While transferring the aqueous phase to a fresh tube, make sure nothing even traces from the interphase or organic phase is transferred along with the aqueous phase. It would be a good idea to sacrifice some aqueous phase instead of transferring traces of other phases.
Step 3: Recover RNA from the aqueous phase by isopropanol-precipitation.
◊ Add 1 volume of ice-cold isopropanol (if the volume of the aqueous phase is ≈400 µL, add 400 µL isopropanol) in the aqueous phase and mix by inverting the tube several times.
◊ Centrifuge at high speed (12,000 × g or more) for 30 min at 4°C.
◊ Discard the supernatant.
Tip:
It is not required to remove the supernatant completely. If there is some supernatant left in this step, it will not harm the next step, which is washing with 75% ethanol.
Precaution:
The pellet of RNA will appear transparent and will be difficult to see. It will also be loosely attached to the bottom of the centrifuge tube. It is possible that while discarding the supernatant, you may lose the RNA pellet, so be careful at this step. So, instead of decanting the supernatant, you can use a micropipette (P1000) to remove the supernatant.
Step 4: Washing of RNA pellet with 75% ethanol.
◊ Dislodge the pellet by flicking the tube with your finger a few times or by vortexing for a few seconds.
◊ Add 500 µL of 75% ethanol and mix by inverting the tube several times.
◊ Centrifuge at high speed (12,000 × g or more) for 15 min at 4°C.
◊ Carefully discard the supernatant without losing the pellet.
◊ Centrifuge the tube for a few seconds to bring the remaining liquid to the bottom of the tube. Use a P100 micropipette to remove it.
◊ Air-dry the pellet for 5 – 10 min by leaving it on the bench with the cap open.
Precaution:
Don’t use a vacuum centrifuge to dry the pellet. It may be difficult to dissolve the pellet if it is too dry.
Step 5: Dissolve the pellet in 30 – 50 µL of nuclease-free water.
Add 30 – 50 µL of nuclease-free water in the pellet and leave it at room temperature for 20 – 30 min with occasionally shaking the tube to dissolve the RNA.
Tip:
If you want to dissolve RNA by incubating it at 60°C for 10 – 15 min, you must add 30 – 50 µL of 0.1 mM EDTA instead of nuclease-free water. Heating RNA in presence of divalent cations leads to hydrolysis of RNA. EDTA chelates divalent cations, thus protecting RNA from hydrolysis.
Optional: You can proceed to determine concentration of RNA and check the quality by gel electrophoresis.
Storage:
RNA can be stored at -20°C (for few months) or – 80°C (for years).
