Analysis of Staphylococcus aureus Gene Expression During PMN Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 110

8. Transfer RNeasy column into a new 2-mL collection tube. Apply 500 μL of
RPE buffer (from RNeasy kit). Wash the column by centrifugation at ≥ 8000 × g

for 60 s. Discard flow-through.
9. To remove residual ethanol, place the RNeasy column in a new 2-mL collection
tube and centrifuge at maximum speed for 2 min.
10. To elute purified RNA, transfer the RNeasy column to a new 2-mL collection
tube, pipet 40 μL of RNase-free water directly onto the silica-gel membrane,
and centrifuge at 8000 × g for 1 min. Run eluted RNA over the same column

again to increase RNA yield.

≥

3.4. DNase Digestion and Clean-Up of S. aureus RNA
DNA contamination is difficult to remove from S. aureus RNA preparations.
The protocol below uses a single on column DNase digestion followed by
two consecutive RNA clean-up assays. The RNeasy silica-membrane removes
DNA; therefore, additional clean-up steps are used to aid in removal of contam-
inating S. aureus DNA.

1. To purified S. aureus RNA, add 60 μL of RNase-free water (total sample volume
should be 100 μL), add 350 μL RLT to the sample, and mix thoroughly by
pipetting.
2. Add 250 μL of ethanol and mix thoroughly by pipetting.
3. Apply the sample to an RNeasy mini column placed in a collection tube (2-mL)
and centrifuge for 60 s at 8000 × g . Discard flow-through.

4. Wash the column by adding 350 μL Buffer RW1 and centrifuging for 60 s at
≥

≥

8000 × g .
5. Add 80 μL of DNase solution. DNase solution contains ˜27 Kunitz U resuspended
in RDD buffer. It is made by adding 10 μL of DNase stock solution (750 Kunitz
U/mL ) to 70 μL RDD buffer.
6. Incubate sample with DNase for 15 min at room temperature.
7. Wash column with 350 μL Buffer RW1 and centrifuge for 60 s at ≥ 8000 × g

and discard flow-through.
8. Transfer the RNeasy column into a new collection tube (2-mL). Wash column
with 500 μL Buffer RPE by centrifugation for 60 s at ≥ 8000 × g , discard

flow-through, and repeat RPE wash.
9. Place the RNeasy column into a new collection tube (2-mL) and centrifuge for
2 min at maximum speed to remove residual ethanol.
10. Elute purified RNA by transferring the RNeasy column to a new 2-mL collection
tube, pipet 40 μL of RNase-free water directly onto the silica-gel membrane,
and centrifuge at 8000 × g for 1 min. Run eluted RNA over the same column

again to increase RNA yield.
11. Repeat RNA clean-up procedure. Follow steps 1-3 and 8-10 (skip DNase steps
4-7 ). To ensure removal of DNA contamination, perform one additional clean-up
step. Although this procedure is time consuming, it results in very high quality

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