Vladamir Motin , University of Texas Medical Branch
Treatment protocol
none
Growth protocol
Grown in custom media and samples collected at 2 hours post temperature shift.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the Ambion mirVana RNA extraction protocol as described by the manufacturer.
Label
CY5
Label protocol
DNA probes for microarray experiments were generated by adding 2 ug of total RNA in a mixture containing 6 ug of random hexamers (Invitrogen), 0.01 M dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amino-allyl-dUTP (aa-dUTP) (Sigma), reaction buffer, and 400 units of SuperScript III reverse transcriptase (Invitrogen) at 42 C degree overnight. The RNA template then was hydrolyzed by adding NaOH and EDTA to a final concentration of 0.2 and 0.1 M, respectively, and incubating at 70 C degree for 15 min. Unincorporated aa-dUTP was removed with a Minelute column (Qiagen). The probe was eluted with a phosphate elution buffer (4 mM KPO4, pH 8.5, in ultrapure water), dried, and resuspended in 0.1 M sodium carbonate buffer (pH 9.0). To couple the amino-allyl cDNA with fluorescent labels, normal human serum-Cy3 or normal human serum-Cy5 (Amersham) was added at room temperature for 1h. Uncoupled label was removed using the Qiagen Minelute column (Valencia, CA).
Vladamir Motin , University of Texas Medical Branch
Treatment protocol
none
Growth protocol
Grown in custom media and samples collected after 2 hours at 26 degrees
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the Ambion mirVana RNA extraction protocol as described by the manufacturer.
Label
CY3
Label protocol
DNA probes for microarray experiments were generated by adding 2 ug of total RNA in a mixture containing 6 ug of random hexamers (Invitrogen), 0.01 M dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amino-allyl-dUTP (aa-dUTP) (Sigma), reaction buffer, and 400 units of SuperScript III reverse transcriptase (Invitrogen) at 42 C degree overnight. The RNA template then was hydrolyzed by adding NaOH and EDTA to a final concentration of 0.2 and 0.1 M, respectively, and incubating at 70 C degree for 15 min. Unincorporated aa-dUTP was removed with a Minelute column (Qiagen). The probe was eluted with a phosphate elution buffer (4 mM KPO4, pH 8.5, in ultrapure water), dried, and resuspended in 0.1 M sodium carbonate buffer (pH 9.0). To couple the amino-allyl cDNA with fluorescent labels, normal human serum-Cy3 or normal human serum-Cy5 (Amersham) was added at room temperature for 1h. Uncoupled label was removed using the Qiagen Minelute column (Valencia, CA).
Hybridization protocol
Aminosilane-coated slides were prehybridized in 5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) (Invitrogen), 0.1% sodium dodecyl sulfate, and 1% bovine serum albumin at 42 C degree for 60 min. The slides then were washed at room temperature with distilled water, dipped in isopropanol, and allowed to dry. Equal volumes of the appropriate Cy3- and Cy5-labeled probes were combined, dried and then resuspended in a solution of 40% formamide, 5x SSC, and 0.1% sodium dodecyl sulfate. Resuspended probes were heated to 95 C degree prior to hybridization. The probe mixture then was added to the microarray slide and allowed to hybridize overnight at 42 C degree. Hybridized slides were washed sequentially in solutions of 1x SSC-0.2% SDS, 0.1x SSC-0.2% SDS, and 0.1x SSC at room temperature, then dried in air.
Scan protocol
Hybridized slides were scanned with an Axon GenePix 4000 scanner
Data processing
Individual TIFF images from each channel were analyzed with TIGR Spotfinder (available at (http://pfgrc.jcvi.org/index.php/bioinformatics.html). Microarray data were normalized by LOWESS normalization and with in-slide replicate analysis using TM4 software MIDAS (available at (http://pfgrc.jcvi.org/index.php/bioinformatics.html). VALUE = log2(QUERY/REF), i.e., the base 2 logarithm of the ratio of QUERY_INTEGRATED_INTENSITY over REF_INTEGRATED_INTENSITY where REF refers to channel 2 (grown at 26 C) and QUERY refers to channel 1 (grown at 37 C).
