(b) FwdAD-AAC (GTTAGATCGGAAGAGCGTAATGATA
CGGCGACCACCGACACTCTTTCCCTACACGACG
CTCTTCCGATCTAAC)
(c) FwdAD-CGA (TCGAGATCGGAAGAGCGTAATGATA
CGGCGACCACCGACACTCTTTCCCTACACGACGC
TCTTCCGATCTCGA)
(d) FwdAD-CTG (CAGAGATCGGAAGAGCGTAATGATA
CGGCGACCACCGACACTCTTTCCCTACACGACGC
TCTTCCGATCTCTG)
(e) FwdAD-GTC (GACAGATCGGAAGAGCGTAATGATA
CGGCGACCACCGACACTCTTTCCCTACACGACGC
TCTTCCGATCTGTC)
(f) FwdAD-GCG (CGCAGATCGGAAGAGCGTAATGATA
CGGCGACCACCGACACTCTTTCCCTACACGACGC
TCTTCCGATCTGCG)
(g) FwdAD-TAA (TTAAGATCGGAAGAGCGTAATGATA
CGGCGACCACCGACACTCTTTCCCTACACGACGC
TCTTCCGATCTTAA)
(h) FwdAD-TCT (AGAAGATCGGAAGAGCGTAATGATA
CGGCGACCACCGACACTCTTTCCCTACACGACGC
TCTTCCGATCTTCT)3 Methods
3.1 RNA Preparation
Using the miRNeasy
Mini Kit (See Note 1)
- Harvest the bacterial cells at the desired time by adding
2 volumes RNAprotect Bacteria Reagent (Qiagen) to 1 volume
bacterial culture. Mix immediately by vortexing for 5 s, and
then incubate at room temperature for 5 min. Next, centrifuge
the cell mixture for 10 min at 5000g. After centrifugation,
decant the supernatant, gently blot the inverted tube on a
paper towel or Kimwipe, and leave inverted for 10 s to remove
as much residual supernatant as possible (seeNote2). The
pellets can then be stored at 15 to 30 C for up to
2 weeks or at 80 C for up to 4 weeks. - Thaw pellets at room temperature, immediately resuspend each
pellet in 1 ml RLT (+βME) (seeNote3) and pipet into a 2 ml
screw cap tube containing 0.1 mm silica beads (seeNote4), and
place on wet ice. Once all samples are prepared and chilled, beat
each sample in beadbeater at maximum rpm for 1 min. Imme-
diately chill the tube on wet ice for 5 min. Repeat as needed;
often another round of bead-beating is needed (seeNote5). - Spin at 4C for 15 min.
- Taking care to leave the beads behind, remove 750μl of the
supernatant and place in a fresh 1.5 ml microfuge tube
182 Charlotte D. Majerczyk