DNase I treat the samples to remove contaminating DNA,
using the Qiagen RNase-free DNase kit. Apply 80 μL of
DNase I: buffer RDD mix (10 μL DNase I, 70 μL RDD buf-
fer, prepared immediately before use) directly onto the col-
umn matrix. Incubate at room temperature for 15 min.
Wash the column with 350 μL RW1 buffer and centrifuge in a
microcentrifuge at 9000 × g for 15 s. Discard the
flow-through.
Wash the column with 500 μL RPE buffer, centrifuge in a
microcentrifuge at 9000 × g for 15 s. Discard the
flow-through.
Pipette 500 μL RPE buffer onto the column matrix and cen-
trifuge at 9000 × g in a microcentrifuge for 2 min. Place the
column into a fresh 2 mL collection tube and centrifuge in a
microcentrifuge for an additional 1 min at 15,000 × g to pre-
vent carry-over of wash buffer.
Transfer the column to a fresh nuclease-free 1.5 mL tube and
add 30 μL RNase-free water directly onto the column matrix.
Incubate at room temperature for 2 min and centrifuge at
9000 × g in a microcentrifuge for 1 min to elute the
RNA. Reapply the eluate to the column, incubate for further
2 min and centrifuge at 9000 × g for 1 min (see Note 10).
Quantify the RNA samples and assess the integrity of the RNA
using the NanoDrop Spectrophotometer and Agilent 2100
Bioanalyzer (or similar) following manufacturers’
instructions.
Store the samples at − 70 °C (see Note 11) or continue with
amplification of the RNA using the Bacteria MessageAmp II
system.
Adjust RNA sample volume to 5 μL with nuclease-free water
(see Note 12). Incubate for 10 min at 70 °C, before placing
on ice for 3 min. Briefly centrifuge, then add 5 μL polyadenyl-
ation master mix (1 μL 10× buffer, 1 μL RNase inhibitor,
0.5 μL ATP, 1 μL PAP, 1.5 μL nuclease-free water) and incu-
bate at 37 °C for 15 min. Place on ice before proceeding
immediately to the next step.
Add 10 μL reverse transcription master mix (1 μL 10× first
strand buffer, 1 μL T7 oligo-dT, 4 μL dNTP mix, 1 μL
ArrayScript reverse transcriptase, 3 μL nuclease-free water),
mix gently by pipetting and incubate at 42 °C for 2 h. Place
the reactions on ice, then centrifuge briefly.
Add 80 μL second strand master mix (10 μL 10× second
strand buffer, 4 μL dNTP mix, 1 μL RNase H, 2 μL DNA
polymerase, 63 μL nuclease-free water), mix by pipetting then
incubate at 16 °C for 2 h. Return to ice, centrifuge briefly.
