Changes in gene expression in the ME during the course of OM in mice was
evaluated using DNA microarrays, as described elsewhere.34 (link) Briefly, WT C57Bl/6:CB F1 hybrid mice (60–90 d old) were purchased from
Jackson Laboratories (Bar Harbor, ME). Twenty mice per time point were
inoculated bilaterally with NTHi strain 3655. The ME mucosae
were harvested at different intervals: 0 (no treatment control), 3 h, 6 h, and
1, 2, 3, 5, and 7 d after NTHi infection. Sham-inoculated (saline) animals
served as additional controls. Total RNA was extracted using TRIzol (Invitrogen,
CA). The RNA quality was assessed using the RNA 6000 Labchip Kit on the Agilent
2100 Bioanalyzer to ensure the integrity of 18S and 28S ribosomal RNA. Reverse
transcription of the mRNA was done using a T7-oligodT primer and T7 RNA
polymerase to generate biotinylated cRNA probes that were hybridized onto two
Affymetrix MU430 2.0 microarrays per time point sample. The procedures were then
duplicated for each time point to obtain a second, independent replication. The
raw data of gene expression levels were median normalized and statistical
differences in gene transcript expression levels were evaluated using a
variance-modeled posterior inference approach (VAMPIRE).35 (link) Individual transcript fold-level changes were visualized using Genespring
(Agilent Technologies, Santa Clara, CA).
evaluated using DNA microarrays, as described elsewhere.34 (link) Briefly, WT C57Bl/6:CB F1 hybrid mice (60–90 d old) were purchased from
Jackson Laboratories (Bar Harbor, ME). Twenty mice per time point were
inoculated bilaterally with NTHi strain 3655. The ME mucosae
were harvested at different intervals: 0 (no treatment control), 3 h, 6 h, and
1, 2, 3, 5, and 7 d after NTHi infection. Sham-inoculated (saline) animals
served as additional controls. Total RNA was extracted using TRIzol (Invitrogen,
CA). The RNA quality was assessed using the RNA 6000 Labchip Kit on the Agilent
2100 Bioanalyzer to ensure the integrity of 18S and 28S ribosomal RNA. Reverse
transcription of the mRNA was done using a T7-oligodT primer and T7 RNA
polymerase to generate biotinylated cRNA probes that were hybridized onto two
Affymetrix MU430 2.0 microarrays per time point sample. The procedures were then
duplicated for each time point to obtain a second, independent replication. The
raw data of gene expression levels were median normalized and statistical
differences in gene transcript expression levels were evaluated using a
variance-modeled posterior inference approach (VAMPIRE).35 (link) Individual transcript fold-level changes were visualized using Genespring
(Agilent Technologies, Santa Clara, CA).
