|
| Status |
Public on May 09, 2016 |
| Title |
37 C grown wild-type versus reference RNA, bio rep 2, tech rep 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Reference RNA
|
| Organism |
Vibrio cholerae |
| Characteristics |
strain: A1552, wild-type
|
| Growth protocol |
V. cholerae was grown aerobically overnight at 37 C in LB broth, then diluted 1:200 in fresh medium, and harvested either at mid-exponential phase at an OD600 of 0.4 or 1 h after shift to 15 C or 25 C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Two ml aliquots of cultures were collected by centrifugation for 2 min at room temperature. Cell pellets were immediately resuspended in 1 ml Trizol reagent (Invitrogen) and stored at –80˚C. Total RNA was isolated from the cell pellets according to the manufacturer’s instructions (Invitrogen). Contaminating DNA was removed by incubating RNA with RNase-free DNase I (Ambion), and an RNeasy Mini kit (QIAGEN) was used to clean up RNA after DNase digestion.
|
| Label |
Cy5
|
| Label protocol |
RNA samples from test and reference samples were used in a reverse transcription reaction as follows: 3 µg of RNA and 5 µg of random hexamers were denatured at 80°C for 8 min and then chilled on ice for 5 min. The reaction mixture, containing first-strand buffer, 0.01 M dithiothreitol, aminoallyl deoxynucleoside triphosphate labeling mix with amino acid-dUTP in a 3 (dTTP):2 (amino acid-dUTP) ratio, and 400 U SuperScript III (Invitrogen), was added to cooled samples and incubated at 42°C for 3 to 4 h. The cDNA-RNA mixture, samples were incubated at 65°C for 10 min in the presence of 100 mM NaOH and 10 mM EDTA. Reactions were neutralized by the addition of 1 M HEPES, pH 7.0, at a final concentration of 0.5 M HEPES. cDNA was purified from the reactions using a PCR purification kit (Qiagen) with phosphate wash and phosphate elution buffers and dried. Cleaned and dried samples were resuspended in freshly prepared 50 mM Na-bicarbonate buffer, pH 9.0. One vial of Cy3/Cy5 fluor was mixed with each sample and incubated for 1 h in the dark at room temperature. Unincorporated dye molecules were removed using PCR purification kit (Qiagen). Dye incorporation and cDNA concentrations were determined by measuring optical densities at 260, 550, and 650 nm.
|
| |
|
| Channel 2 |
| Source name |
RNA from cells grown at 37 C
|
| Organism |
Vibrio cholerae |
| Characteristics |
strain: A1552, wild-type
growth protocol: grown at 37 C
|
| Growth protocol |
V. cholerae was grown aerobically overnight at 37 C in LB broth, then diluted 1:200 in fresh medium, and harvested either at mid-exponential phase at an OD600 of 0.4 or 1 h after shift to 15 C or 25 C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Two ml aliquots of cultures were collected by centrifugation for 2 min at room temperature. Cell pellets were immediately resuspended in 1 ml Trizol reagent (Invitrogen) and stored at –80˚C. Total RNA was isolated from the cell pellets according to the manufacturer’s instructions (Invitrogen). Contaminating DNA was removed by incubating RNA with RNase-free DNase I (Ambion), and an RNeasy Mini kit (QIAGEN) was used to clean up RNA after DNase digestion.
|
| Label |
Cy3
|
| Label protocol |
RNA samples from test and reference samples were used in a reverse transcription reaction as follows: 3 µg of RNA and 5 µg of random hexamers were denatured at 80°C for 8 min and then chilled on ice for 5 min. The reaction mixture, containing first-strand buffer, 0.01 M dithiothreitol, aminoallyl deoxynucleoside triphosphate labeling mix with amino acid-dUTP in a 3 (dTTP):2 (amino acid-dUTP) ratio, and 400 U SuperScript III (Invitrogen), was added to cooled samples and incubated at 42°C for 3 to 4 h. The cDNA-RNA mixture, samples were incubated at 65°C for 10 min in the presence of 100 mM NaOH and 10 mM EDTA. Reactions were neutralized by the addition of 1 M HEPES, pH 7.0, at a final concentration of 0.5 M HEPES. cDNA was purified from the reactions using a PCR purification kit (Qiagen) with phosphate wash and phosphate elution buffers and dried. Cleaned and dried samples were resuspended in freshly prepared 50 mM Na-bicarbonate buffer, pH 9.0. One vial of Cy3/Cy5 fluor was mixed with each sample and incubated for 1 h in the dark at room temperature. Unincorporated dye molecules were removed using PCR purification kit (Qiagen). Dye incorporation and cDNA concentrations were determined by measuring optical densities at 260, 550, and 650 nm.
|
| |
|
| |
| Hybridization protocol |
Dye-coupled test samples were mixed with the corresponding reference sample and dried. Amine silane slides containing 70-mer oligonucleotides, representing most of the open reading frames present in the V. cholerae N16961 genome, were UV cross-linked at 250 mJ and prehybridized in a solution containing 5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate, and 1% bovine serum albumin for at least 45 min at 42°C. The hybridization mix was prepared by resuspending dried samples in a solution containing 15 µg salmon sperm DNA, 15 µg tRNA, 3x SSC, and 0.1% sodium dodecyl sulfate. Concentrated hybridization mix was applied to the slides and incubated at 65°C for 12 to 20 h.
|
| Scan protocol |
Slides were washed, dried, and scanned using an Axon scanner to determine the fluorescence in each open reading frame-specific spot. The raw data were obtained by using the software package GenePix 4.1 (Axon).
|
| Description |
Reference RNA was harvested from wild-type strain. Each sample contains two technical replicates within the same .gpr file, becasue each oligonucleotide was printed twice on the same slide.
|
| Data processing |
Normalized signal ratios were obtained with LOWESS print-tip normalization using the Bioconductor packages (http://www.bioconductor.org) in R environment. Differentially regulated genes were determined (with three biological and two technical replicates for each data point) with the significance analysis of microarrays (SAM) package using twofold differences in gene expression and a 3% false discovery rate as a cutoff value.
|
| |
|
| Submission date |
May 05, 2016 |
| Last update date |
May 09, 2016 |
| Contact name |
Loni Townsley |
| E-mail(s) |
townsley@unc.edu
|
| Organization name |
University of North Carolina
|
| Department |
Biology
|
| Street address |
250 Bell Tower Dr Genome Sciences Building #4144
|
| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599 |
| Country |
USA |
| |
|
| Platform ID |
GPL20916 |
| Series (1) |
| GSE81158 |
Vibrio cholerae temperature shift microarray analysis |
|
