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tion, washing the universals with GTC solution to ensure all
bacilli are recovered. Spin in a single universal tube per sample
at 1800 × g in a benchtop centrifuge for 20 min and remove
the supernatant.- Resuspend the pellet in 1 mL TRIzol and transfer the suspen-
sion to a 2 mL screw-capped tube containing 0.5 mL of
0.1 mm silica beads. Wash the universal tube with an addi-
tional 200 μL TRIzol to recover all bacilli in 1.2 mL final vol-
ume (see Notes 6 and 7 ). - Lyse the bacteria using a reciprocal shaker at speed 6.5 for
45 s, then incubate at room temperature for 10 min. - Add 200 μL chloroform to each sample, vortex for 30 s, then
incubate at room temperature for 10 min to partition the
aqueous and phenolic phases. Centrifuge at 15,000 × g in a
microcentrifuge for 15 min at 4 °C. - Transfer the aqueous phase to a new 1.5 mL tube, add an
equal volume of chloroform and centrifuge at 15,000 × g in a
microcentrifuge for 15 min at 4 °C. - Transfer the aqueous phase to a fresh nuclease-free 1.5 mL
tube, add 0.8 volume of isopropanol and mix by inverting.
Incubate overnight at − 20 °C to precipitate the nucleic acids
(see Note 8). - Centrifuge the samples in a microcentrifuge at 15,000 × g for
20 min at 4 °C to pellet the nucleic acid (see Note 9). - Remove the supernatant carefully by pipetting and wash the
pellet with 500 μL cold 70% ethanol. Centrifuge in a micro-
centrifuge at 15,000 × g for 15 min at 4 °C. - Remove the ethanol by pipetting, respin the tubes briefly and
remove any additional liquid. - Air-dry the pellet for 5–10 min at room temperature and
resuspend in 100 μL RNase-free water. Store briefly on ice and
proceed to RNA cleanup using RNeasy Mini Columns, with
buffers prepared and stored according to manufacturer’s
instructions. - Add 350 μL RNeasy RLT buffer to each 100 μL sample and
mix thoroughly by pipetting. - Add 250 μL 100% ethanol, mix thoroughly by pipetting and
apply immediately to an RNeasy Mini Column placed in a
2 mL collection tube. Centrifuge in a microcentrifuge at
9000 × g for 15 s, discard the flow-through. - Apply 350 μL RNeasy RW1 buffer to the column, centrifuge
in a microcentrifuge at 9000 × g for 15 s and discard the
flow-through.
Leticia Muraro Wildner et al.