RNA extraction from Actinobacteria
Isolation of high quality RNA from actinobacterial species
This protocol can be used to isolate high quality RNA from actinobacterial strains for downstream applications including total RNA sequencing and RT-qPCR. Strains can be grown in liquid media, or on solid agar media before extraction. This protocol is an optimised version of the QIAGEN RNeasy mini kit extraction protocol and you will need to acquire this kit in order to proceed.
- All pipette tips, eppendorfs and other tubes should be autoclaved twice before use, unless you are sure of that they are RNase free.
- Treat solutions that are not supplied in the QIAGEN RNeasy mini kit and that are autoclavable (i.e the sterile water) with Diethyl pyrocarbonate (DEPC) to remove RNases. For DEPC treatment add 0.1% DEPC to the solution, then incubate overnight at 37°C before autoclaving. For any glassware that needs treating, fill with 0.1% DEPC in sterile water, shake and incubate overnight at 37°C then autoclave and pour away the liquid.
- Clean all surfaces, gloves and pipettes with RNaseZap (or a similar product that eliminates RNases) before carrying out the protocol. Change gloves regularly.
Collection of cellular material
- Collect cellular material (if strains are grown in liquid, pellet the cellular material by centrifugation in microcentrifuge tubes. If solid agar conditions are used for growth, it can be helpful to grow strains on sterile cellophane discs that lie on top of the agar; cell material can then be easily scraped off with a flame-sterilised spatula).
- Quickly snap freeze collected cellular material in liquid nitrogen immediately after sampling to avoid degradation of RNA. Store at -80°C until further processing.
- Manually grind cell pellets to a fine powder using a hand-held micropestle. Samples should be placed in a rack on dry ice and the rack should be cooled with liquid nitrogen during this process.
- Add 700 µl of Qiagen buffer RLT (supplemented with 1% betamercapterethanol) to ground samples without allowing them thaw. Qiagen buffer RLT is a lysis buffer supplied with the QIAGEN RNeasy mini kit.
- Vortex thoroughly to mix. Centrifuge the sample briefly to pellet if too dense to pipette.
- Add the supernatent to a QIAshredder column (Qiagen) and centrifuge for 2 minutes at 13000 rpm. This step ensures homogenisation of the sample.
- Collect the resulting flow through from the column (avoid taking up any pellet that has formed on the side of the collection tube) and add 700 µl of acid phenol chloroform to this. Mix by turning slowly for one minute.
- Incubate the samples for 3 minutes on the benchtop at room temperature, then centrifuge for 20 minutes in a benchtop centrifuge at 13000 rpm.
- Collect the upper phase (avoid taking up any of the separating layer) and mix with a 50% volume of 96% ETOH (made up with RNase free, DEPC treated water). Transfer the mixture to a Qiagen mini spin column with collection tube (supplied with the RNeasy mini kit).
- The remainder of the protocol uses the RNeasy mini kit protocol (from step 3), including on-column digestion with DNase I. In the final step, elute the RNA in 50 µl of RNase free water. RNA yield can be enhanced by incubating the water for 5 minutes at room temperature on the column membrane before centrifuging to collect.
- It is advised to subject RNA to a further round of DNase treatment using the Invitrogen TURBO DNase kit- it is best to do this immediately after elution, instead of freeze-thawing the RNA sample later. For this, add 5 µl of 10X buffer and 2 µl of TURBO DNase (both in the TURBO DNase kit) to 50 µl of RNA and incubate at 37°C for 25 minutes. Remove the DNase by following the Qiagen RNeasy mini kit on-column clean up protocol. Elute the RNA in 50 µl of RNase free water.
- The quantity and purity of RNA samples can be checked using a nanodrop spectrophotometer and/or Qubit RNA HS or BS assay kit (Invitrogen). An Experion bioanalyser with a prokaryotic RNA standard sensitivity analysis kit (Bio-Rad, California, USA) can be used to assess RNA integrity.
If very small quantities of RNA are expected from samples the QIAGEN RNeasy micro kit could be used to enhance yield. Similarly, if larger quantities of RNA are expected the QIAGEN RNeasy midi kit can be used with the same protocol.
Protocol adapted from the QIAGEN RNeasy mini kit by Dr Sarah Worsley, Postdoc at the University of East Anglia.