Overview:
- Cell lines are very useful tools for life science research. Isolation of total RNA from cell lines is often needed especially when analyzing and comparing gene expression in different sets of experimental conditions.
- A major challenge in RNA isolation is to protect the RNA from endogenous RNases during the lysis of cells.
- Trizol, a monophasic solution of GITC and Phenol, not only quickly solubilizes the cells but also simultaneously and efficiently inactivates endogenous RNases, leading to the recovery of intact RNA from cells.
- Cell monolayer allows Trizol easy access to all cells without the requirement of vigorous and harsh homogenization steps. This makes the solubilization process easy and efficient as compared to the solubilization of tissues.
- The protocol described here is suitable for the isolation of total RNA using Trizol from almost any adherent cell culture including both primary and tumor cell culture. Several samples can be processed simultaneously.
- 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:
Cell scraper
Trizol
Chloroform
Nuclease-free water
Isopropyl alcohol
75% Ethanol
Equipment and disposables
Micropipette
Micropipette tips
Refrigerated centrifuge
1.5 or 2 ml microcentrifuge tubes
Starting material:
Exponentially-growing adherent cells (60 – 70 % confluent adherent culture) in 6 cm dish/T25 flask
Prior to start
♦ Examine the culture under an inverted phase-contrast microscope. Make sure that they fulfill the requirements of your experiment and there is no visible contamination.
♦ Set the temperature of the centrifuge to 4°C.
Note:
Since culture dishes are to be used for RNA isolation, there is no need to maintain sterile conditions.
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: Remove and discard culture medium from the dish and lyse the cell monolayer in Trizol.
◊ Take out the culture vessel from the incubator and place it inside the fume hood.
◊ Slightly tilt the culture dish and remove and discard the medium using a pipette or an aspirator.
◊ Quickly add 1 ml Trizol and swirl or tilt the culture dish in all directions to spread Trizol over the cell surface.
◊ Now with a help of a scraper, collect all cell lysate to one side of the dish.
◊ Pipette lysate up and down several times to lyse the cells completely.
◊ Transfer lysate to a 1.5 ml microcentrifuge tube using a P1000 micropipette. Incubate at room temperature for 2 – 3 min.
Precautions:
◊ You must add Trizol quickly after removing the culture medium from the dish. Don’t let the cell monolayer dry.
◊ Washing cell monolayer with ice-cold PBS is not recommended as washing can contribute to mRNA degradation.
◊ Try to remove the culture medium as much as possible from the dish. The final lysate volume should not exceed 10% of the volume of Trizol used for preparing lysate i.e., in our case the final volume of lysate should be less than 1.1 ml.
Note:
Trizol is a monophasic solution of GITC and acidic phenol. Both reagents are highly efficient at lysing the cells and denaturing RNases.
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 as interphase by some)
◊ Transfer the upper clear aqueous phase to a fresh 1.5 ml centrifuge tube.
◊ Save precipitate (the interphase) and organic phase if you want to isolate DNA and protein, otherwise discard the tube.
Tips:
◊ 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.
Precautions
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.
Precautions
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 it with your finger a few times or by vortexing briefly 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 the 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).
