RNA from R. palustris wild type/fixK mutant grown microaerobically
Growth protocol
Cells were grown on PM medium in the dark. Cultures were continuously bubbled with a gas mix (94%N2/6%O2). Flasks were placed on a magnetic stirrer turning at ~100 rpm to allow proper mixing of the cultures. The culture medium was equilibrated with the desired gas mixture for at least 12 hours before inoculation. Cells were harvested at OD660 of 0.25-0.35. Cells were harvested at OD660 of 0.25-0.35.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated using a modification of the RNeasy midi kit (Qiagen, Chatsworth, CA).
Label
Cy3
Label protocol
Labeled cDNA was prepared by direct incorporation of either the Cy3-dCTP or Cy5-dCTP fluorophore (Amersham Biosciences) during a first-strand reverse transcription reaction. Each 45 µl reaction contained 12 µg of total RNA, 13.5 µg of random primers (Invitrogen), 9 µl of 10 mM DTT, 0.5 mM each of dATP, dGTP, and dTTP, 0.2 mM dCTP, 40 U RNasin (Promega, Madison, WI), 3 µl of 1 mM Cy3- or 1 mM Cy5-dCTP, and 600 U of SuperScript II reverse transcriptase (Invitrogen). After a 2 hour incubation at 42ºC, 15 µl of 0.5 M EDTA (pH 8.0) and 15 µl of 1M NaOH were added to the sample, and incubation was continued at 65ºC for 30 minutes. The sample was then neutralized by adding 30 µl of 3M sodium acetate (pH 5.2) and 45 µl of H2O to bring the volume to 150 µl. The labeled cDNA was purified with the QIAquick PCR purification kit (Qiagen, Chatsworth, CA). The labeling efficiency was calculated by measuring the A260 and either A550 for Cy3 incorporation or A650 for Cy5 incorporation
RNA from R. palustris wild type/fixK mutant grown microaerobically
Growth protocol
Cells were grown on PM medium in the dark. Cultures were continuously bubbled with a gas mix (94%N2/6%O2). Flasks were placed on a magnetic stirrer turning at ~100 rpm to allow proper mixing of the cultures. The culture medium was equilibrated with the desired gas mixture for at least 12 hours before inoculation. Cells were harvested at OD660 of 0.25-0.35. Cells were harvested at OD660 of 0.25-0.35.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated using a modification of the RNeasy midi kit (Qiagen, Chatsworth, CA).
Label
Cy5
Label protocol
Labeled cDNA was prepared by direct incorporation of either the Cy3-dCTP or Cy5-dCTP fluorophore (Amersham Biosciences) during a first-strand reverse transcription reaction. Each 45 µl reaction contained 12 µg of total RNA, 13.5 µg of random primers (Invitrogen), 9 µl of 10 mM DTT, 0.5 mM each of dATP, dGTP, and dTTP, 0.2 mM dCTP, 40 U RNasin (Promega, Madison, WI), 3 µl of 1 mM Cy3- or 1 mM Cy5-dCTP, and 600 U of SuperScript II reverse transcriptase (Invitrogen). After a 2 hour incubation at 42ºC, 15 µl of 0.5 M EDTA (pH 8.0) and 15 µl of 1M NaOH were added to the sample, and incubation was continued at 65ºC for 30 minutes. The sample was then neutralized by adding 30 µl of 3M sodium acetate (pH 5.2) and 45 µl of H2O to bring the volume to 150 µl. The labeled cDNA was purified with the QIAquick PCR purification kit (Qiagen, Chatsworth, CA). The labeling efficiency was calculated by measuring the A260 and either A550 for Cy3 incorporation or A650 for Cy5 incorporation.
Hybridization protocol
Prior to performing hybridizations, the array slides were incubated in prehybridization buffer at 65oC for 45 min in prehybridization buffer (3X SSC [1X SSC is 0.15M NaCl plus 0.015M sodium citrate], 0.3% sodium dodecyl sulfate (SDS), and 1% bovine serum albumin). The two labeled cDNA samples to be compared were mixed, and 20X SSC (final concentration ,3X), 10mg/ml salmon sperm DNA (Invitrogen; final concentration, 0.8 mg/ml), and 5% SDS (final concentration 0.3%) were added to the sample. This hybridization mixture was divided into two tubes, incubated at 95oC for 3 minutes, and the mixture in each tube was applied to prehybridized slides that had been covered with Lifterslips (Erie Scientific Company, NH). The slides were assembled with hybridization chambers (Corning, NY) and submerged in a 65oC water bath for 14-16 hours. After hybridization the slides were washed with 1X SSC and 0.2% SDS for 5 minutes, 0.1X SSC and 0.2% SDS for 5 minutes, and 0.1 SSC for 5 minutes. The slides were dried by centrifugation (200 X g for 5 minutes).
Scan protocol
Slides were scanned with a ScanArray 4000XL scanner (Perkin-Elmer, Boston, MA). Images (Cy3 and Cy5) were captured as TIFF files.
Description
The software package IcDNA was used for data normalization and assessment of the statistical confidence intervals of gene expression. Two calibration experiments and three comparative experiments using RNA from three separately grown cultures (three biological replicates) with duplicate slides each (except for one of the calibration experiments ; 9 slides total) were used to generate each data set.
Data processing
Images (Cy3 and Cy5) were captured as TIFF files and were analyzed with image preocessing software ImaGene version 5.6 (BioDiscovery, Inc., El Segundo, CA).
