RNA Isolation and Macroarray Protocol
Growth and RNA Isolation
The prototrophic Escherichia coli K-12 laboratory strain was grown
with orbital shaking in M63 minimal medium containing 0.4% glycerol as sole
carbon source at 37° C . When the bacteria reached an A600 of 0.2, the
culture was split into two equal volumes. Sialic acid (N-acetylneuraminic
acid, 1 mg/ml final concentration) was added to one culture and both cultures
were allowed to grow for an additional 40 minutes. The flasks were removed
from the incubator and 10 ml of each culture were added to 20 ml of RNA Protect
(Qiagen). RNA was purified using the Qiagen RNAeasy kit according to manufacturer’s
instructions, including treatment with DNase. The integrity of the RNA was
checked by running a 5-µg sample on an agarose gel and staining with
EtBr.
RNA from each sample (1 µg) was mixed with 4 µl of E. coli
cDNA labeling primers (Sigma Genosys C5603). This mixture was heated to 94°C
for 2 min and then ramped to 42°C over 20 min in a thermal cycler to allow
primer annealing. cDNA was prepared by the addition of dATP, dGTP, dTTP (333µM),
[a-33P] dCTP (20 mCi with a specific activity of 2,000-3,000 Ci/mmol), 50
Units of AMV Reverse Transcriptase (Sigma Genosys) and Reverse Transcriptase
Buffer. This mixture was incubated for 3 h at 42°C. Unincorporated nucleotides
were removed by purification over a SephadexÔ G-25 spin column (Sigma
Genosys, PRSP0001).
Two Panorama™ E. coli Gene Array membranes (Catalog number
G1666) were prepared by rinsing in 2 x SSPE for 5 minutes. The membranes were
prehybridized with Hybridization Solution (PR485) at 65°C for 2 h. The
labeled cDNA was mixed with 3 ml Hybridization Solution and heated to 95°C
for 10 min, to denature the cDNA. The prehybridization solution was removed
and replaced with the labeled cDNA in Hybridizaiton Solution. Hybridization
was carried out at 65°C, mixing, overnight. The membranes were washed
with Wash solution (0.5x SSPE, 0.2% SDS) as directed in the Panorama protocol.
The array was exposed to a phosphoimager screen for 2 days and scanned on
an Amersham Storm™ or Typhoon™ imager. The data was analyzed using
ArrayVision™ software from Imaging Research.
The growth, isolation of RNA, preparation of cDNA, and macroarray of wild
type Escherichia coli versus a nanR null mutant was carried
out as described above, with the exception that both strains were grown in
M63 minimal medium containing 0.4% glycerol as sole carbon source.
Growth on glycerol with or without sialic acid induction
