containing 50μlRLT+βME. Centrifuge the tube at max speed
for 30 s. Then, transfer 750μl to a fresh 2 ml microfuge tube.
- Add 1.125 ml (1.5volume) of 100% ethanol, vortex, and
apply 700μl of the sample to the RNeasy column. Centrifuge
for 15 s at 14,000 rpm (17,000g) at room temperature,
discard the flow-through, and repeat using the remainder of
the sample in 700μl increments.
- Add 700μl Buffer RWT to the RNeasy column, centrifuge for
30 s at 14,000 rpm (17,000g), and discard the flow-
through. Transfer the RNeasy Mini column into a new 2 ml
collection tube and add 500μl Buffer RPE to the RNeasy Mini
column. Centrifuge for 30 s at 14,000 rpm (17,000g), and
discard the flow-through. Add another 500μl Buffer RPE to
the RNeasy Mini column and centrifuge for 2 min at
14,000 rpm (17,000g). Transfer the RNeasy Mini column
into new 1.5 ml microfuge tube and centrifuge for 1 min at
14,000 rpm (17,000g). Next, transfer the RNeasy Mini
column into another new 1.5 ml microfuge tube. Open the lid
of the RNeasy Mini column and let sit on your bench for
2.5 min to remove any residual ethanol.
- Pipet 30μl RNase-free water directly onto the Mini column
silica-gel membrane, let the column stand for 1 min, and
centrifuge for 1 min at 14,000 rpm (17,000 g). Pipet
another 30μl RNase-free water onto the silica-gel membrane,
let the column stand for 1 min, and centrifuge for 1 min at
14,000 rpm (17,000g). The RNA should be stored at
80 C.
- Determine the RNA concentration using a spectrophotometer.
- Confirm that the RNA is not degraded. One option is to run
the RNA on an agarose gel. To prepare an RNA gel, wipe all
electrophoresis parts and the gel comb with RNAase AWAY
Decontamination Reagent. In a clean beaker, prepare a 1.5%
(wt/vol) agarose gel by melting 1.5 g agarose in 100 ml 1
NorthernMax-Gly Gel Prep Running buffer. Let gel solidify,
remove comb, place in gel box, and submerge in 1
NorthernMax-Gly Gel Prep Running buffer. To prepare the
RNA for electrophoresis, pipet 1 μg of RNA sample in
RNase-free 0.2 ml tube, add RNase-free water to bring volume
to 6μl, add 6μl of Glyoxal Sample Loading Dye (already
contains ethidium bromide), and heat the sample at 50C for
30 min. Allow sample to cool to room temperature and load
12 μl of sample per well. Run electrophoresis at 50–100 V and
observe gel by UV light to confirm the presence of two distinct
bands of RNA (23S, 16S) and potentially minor species
(including 5S). Check that your sample has limited degradation
by ensuring that the bands are crisp and without smears.
RNAseq of QS Regulons 183
