- Cell lines are a very useful tool 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.
- RNA isolation from suspension cell culture requires harvesting cells by centrifugation. Harvested cells are lysed in Trizol. Cell lysate is subjected to phase separation by adding chloroform. RNA is recovered from aqueous phase using isopropanol precipitation. Precipitated RNA is dissolved in water and stored at -20°C or -80°C.
- The protocol described here is suitable for the isolation of total RNA using Trizol from almost any suspension 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.
Reagents and solutions:
Equipment and disposables
1.5 or 2 ml microcentrifuge tubes
Exponentially-growing cell suspension (≈ 70 % confluent culture)
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.
Since culture dishes are to be used for RNA isolation, there is no need to maintain sterile conditions.
♦ 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.
Step 1: Harvest cells by centrifugation and lyse them in Trizol.
◊ Shake the culture to suspended the cells uniformly. Transfer cell suspension (correspond to 5- 10 x 106 cells) to a 15 ml centrifuge and harvest cells by centrifugation at room temperature for 5 – 10 min at 250 × g (1000 – 1500 rpm for Eppendorf™ 5804 Series benchtop centrifuge).
◊ Carefully decant the supernatant, and resuspend the cells in the remaining liquid in the tube.
◊ Quickly add 1 ml Trizol and pipette (use P1000) 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.
If possible, count the cell number to know the initial number of cells taken for RNA isolation.
◊ 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 after centrifugation. 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.
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 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.
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.
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.
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.
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.
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.
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.
RNA can be stored at -20°C (for few months) or – 80°C (for years).