strains were cultured in GAM (Gifu Anaerobic Medium)
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using hot-phenol method
Label
Cy5
Label protocol
15 µg of total RNA from was mixed with 2 µl of random primers (d(N)6, d(N)9, 0.5 µg/µl each, Takara, Japan) and adjusted to 22 µl with water. The solution was kept at 65°C for 5 min to denature the RNA. The annealing reaction was performed at 42°C for 5 min. The premix solution (18 µl) was mixed into RNA solution, and reverse transcription was performed at 42 °C for 60 min. The premix solution was a mixture of 8 µl of x5 power script reaction buffer, 4 µl of dNTPs (5 mM dATP, 5 mM dCTP, 5 mM dGTP, 2 mM dTTP, 3 mM amino-allyl dUTP (Sigma, USA)), 4 µl of 0.1 M DTT, 0.1 µl of RNase inhibitor (TakaRa, Japan), and 2 µl of Powerscript reverse transcriptase (BD Biosciences, USA). After incubation, 5 µl of 0.5M EDTA (pH 8.0) and 10 µl of 1 N NaOH were mixed, and the mixture was incubated at 65°C for 30 min to stop the reaction and remove the RNA templates, followed by the addition of 25 µl of 1 M Tris-Cl (pH 7.5) for neutralization. The mixture was transferred to a Microcon-30 filter (Millipore, USA) and centrifuged. The filter was washed with 100 µl of water 4 times, and the sample was eluted in 25 µl of water. The solution was completely dried, re-suspended in 10 µl of 50 mM NaCO3, and incubated at room temperature (RT) for 3 min. Ten microliters of Cy3 or Cy5 (GE Healthcare, USA) was mixed and incubated at RT for 60 min in a dark place. Ten microliters of Na2OH (Sigma) was added to remove un-reacted Cy-dye. Finally, the labeled product was purified by a QIAquick column (Qiagen, Germany).
strains were cultured in GAM (Gifu Anaerobic Medium)
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using hot-phenol method
Label
Cy3
Label protocol
15 µg of total RNA from was mixed with 2 µl of random primers (d(N)6, d(N)9, 0.5 µg/µl each, Takara, Japan) and adjusted to 22 µl with water. The solution was kept at 65°C for 5 min to denature the RNA. The annealing reaction was performed at 42°C for 5 min. The premix solution (18 µl) was mixed into RNA solution, and reverse transcription was performed at 42 °C for 60 min. The premix solution was a mixture of 8 µl of x5 power script reaction buffer, 4 µl of dNTPs (5 mM dATP, 5 mM dCTP, 5 mM dGTP, 2 mM dTTP, 3 mM amino-allyl dUTP (Sigma, USA)), 4 µl of 0.1 M DTT, 0.1 µl of RNase inhibitor (TakaRa, Japan), and 2 µl of Powerscript reverse transcriptase (BD Biosciences, USA). After incubation, 5 µl of 0.5M EDTA (pH 8.0) and 10 µl of 1 N NaOH were mixed, and the mixture was incubated at 65°C for 30 min to stop the reaction and remove the RNA templates, followed by the addition of 25 µl of 1 M Tris-Cl (pH 7.5) for neutralization. The mixture was transferred to a Microcon-30 filter (Millipore, USA) and centrifuged. The filter was washed with 100 µl of water 4 times, and the sample was eluted in 25 µl of water. The solution was completely dried, re-suspended in 10 µl of 50 mM NaCO3, and incubated at room temperature (RT) for 3 min. Ten microliters of Cy3 or Cy5 (GE Healthcare, USA) was mixed and incubated at RT for 60 min in a dark place. Ten microliters of Na2OH (Sigma) was added to remove un-reacted Cy-dye. Finally, the labeled product was purified by a QIAquick column (Qiagen, Germany).
Hybridization protocol
Hybridization solution (25 μl of Cy-dye-labeled cDNA solution, 10 μl of x20 SSC, 3 μl of water, and 2 μl of 10% SDS) was poured onto the slide glass and covered with a cover-glass with a gap (Matsunami Glass). The slide was inserted into the hybridization chamber, and hybridization was performed at 60 °C overnight.
Scan protocol
Scanned on an FLA-8000 scanner (Fuji Film, Japan). The density of the scanned spots was measured by ArrayVision software (Imaging Research, USA)
Data processing
Normalized by the median of the raw intensities for all spots in each control and sample. LOWESS normalization was performed. GeneSpring software was used.
