Overview:
- Escherichia coli (E. coli) is one of the well characterized bacteria and is extensively used in both applied and basic research.
- E. coli is Gram-negative bacteria. Unlike mammalian cells, E. coli cells are surrounded by a cell wall that protects it from harsh conditions. This also poses a problem while lysing the bacterial cells for RNA isolation.
- Although Trizol can lyse the E. coli cells, some supplementary reagents such as “Max Bacterial Enhancement Reagent” are designed to increase the efficiency of the lysis process, resulting in high yield of RNA.
- The protocol described here is suitable for the isolation of total RNA using Trizol from both Gram-positive and Gram-negative bacteria.
- 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.
- The protocol described here uses “ThermoFisher Scientific’s TRIzol® Max™ Bacterial RNA Isolation Kit” to isolate total RNA from E. coli.
Requirements:
Reagents and solutions:
Trizol
Max Bacterial Enhancement Reagent (see product details)
Chloroform
Isopropyl alcohol (Ice-cold)
Nuclease-free water
75% ethanol (Ice-cold)
Water bath/heat block set at 95°C
Equipment and disposables
Micropipette
Micropipette tips
Refrigerated centrifuge
1.5 or 2 ml microcentrifuge tubes
Starting material:
Exponentially growing E. coli culture (OD600: ≈0.6)
Prior to start
♦ Set the temperature of the centrifuge to 4°C.
♦ Set the temperature of water bath/heat block to 95°C
Note:
Since culture is 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: Harvest bacterial cells by centrifugation and lyse cells in Max reagent/Trizol.
◊ Transfer 1 – 1.5 ml E. coli culture to a 1.5 ml microcentrifuge tube.
◊ Centrifuge the tube at room temperature for 5 min at 6000 × g or 1 min at 12000 × g.
◊ Quickly decant the supernatant after centrifugation and Keep the vial inverted with lid open on a paper towel to let the remaining medium out from the vial.
◊ Vortex the vial for 15 seconds to dislodge the pellet.
◊ Add 200 µL prevarmed (95°C) Max reagent and heat the tube 95°C for 4 min.
◊ Following incubation, vortex the tube for 30 sec to mix the content.
◊ Add 1 ml Trizol and mix by vortexing for 30 sec.
◊ Incubate at room temperature for 5 min.
Precautions:
◊ Centrifugation speed must be sufficient enough to harvest all cells but also generate a pellet which is easy to resuspend. Too high centrifugation speed generates a tight pellet that can not be resuspended easily.
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)
◊ 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.
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 along the aqueous phase.
Step 3: Recover RNA from the aqueous phase by isopropanol-precipitation.
◊ Add 1 volume of ice-cold isopropanol (e.g., 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. 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 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).
