|
| Status |
Public on Feb 11, 2008 |
| Title |
Comparison -2.5°C versus 39°C - Replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
E. sibiricum 255-15 grown at -2.5°C labeled with Cy5 (red)
|
| Organism |
Exiguobacterium sibiricum 255-15 |
| Characteristics |
strain 255-15 grown at -2.5°C
|
| Treatment protocol |
After growing the cells in agar at the four temperatures, a colony from each plate was transferred to tubes containing 5 ml 1/2 TSB and grown in its respective temperatures until an optical density at 600 nm (OD600) of 1.0 was attained. Then 1 ml of this culture was used to inoculate 100 ml of 1/2 TSB in a Nephlo Flask (Belco) for the experiment.
|
| Growth protocol |
All the cells for the DNA microarray experiments came from the same E. sibiricum 255-15 frozen stock that was used for the genome sequencing. All experiments were performed by first plating the cells in 1/2 Tryptic Soy Agar (TSA) and then transferred to 1/2 Tryptic Soy Broth (TSB) twice. In total, six samples were grown independently at 39°C, 28°C, 10°C and -2.5°C. The cells were acclimated by growth at 39°C, 28°C, 10°C and -2.5°C. For the growth at 39°C and 28°C the plates were incubated overnight. At 10°C, the plates were incubated for 3 to 4 days. At -2.5°C the plates were transferred three times to new plates to acclimate the cells to lower temperature as follows: the first plates were incubated overnight at 22°C followed by incubation at 4°C for 3 days and then for 2-3 weeks at -2.5°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The samples were incubated until reaching mid-log growth (0.1 < OD600 < 0.3) when 100 ml of RNAlater (Ambion, Austin, Texas) was added at the same temperature as the grown cells. Cells were pelleted by centrifugation at 5,000 X g for 20 min at 4°C and resuspended in 1 ml of RNAlater, transferred to a 1.5 ml microcentrifuge tube, and re-pelleted at 5,000 X g at 4°C for 10 min.The cells were re-suspended in 100 ul of RNase-free 3 mg ml-1 Lysozyme in TE buffer pH 8 (50 mM Tris-Cl and 1 mM EDTA) by vortexing and then incubated at room temperature for at least 20 min or until the pellet cleared. The RNA was then isolated using the RNeasy mini Prep kit (Qiagen) according to the manufacturer's instructions; the step of DNase digestion was included. The resulting RNA was checked by denaturing agarose gel electrophoresis for DNA contamination and for the presence and integrity of the rRNA bands. The amount of RNA was quantified using a UV-spectrophotometer at OD260.
|
| Label |
Cy5
|
| Label protocol |
Amino-allyl labeling was performed as adapted from a protocol of The Institute for Genomic Research (TIGR) (http://www.tigr.org/tdb/microarray/protocolsTIGR.shtml). Briefly, 10 ug of total RNA was used to synthesize cDNA overnight at 42°C using 0.5 mM of Random Hexamer Primers (Invitrogen, Carlsbad, CA), 3:2 ratio of 5-(3-amino-allyl)-dUTP and dTTP (Ambion), and Superscript II reverse transcriptase (Invitrogen), and subsequently labeled by coupling reactive Cy5 or Cy3 fluorophores (Amersham, Piscataway, NJ) to the amino-allyl groups. Purification after cDNA synthesis and chemical coupling were performed using QiaQuick PCR purification columns (Qiagen) as described in TIGR protocol. The quantity of labeled cDNA and the fluorophore incorporation efficiency were determined by using UV-visible spectrophotometry.
|
| |
|
| Channel 2 |
| Source name |
E. sibiricum 255-15 grown at 39°C labeled with Cy3 (green)
|
| Organism |
Exiguobacterium sibiricum 255-15 |
| Characteristics |
strain 255-15 grown at 39°C
|
| Treatment protocol |
After growing the cells in agar at the four temperatures, a colony from each plate was transferred to tubes containing 5 ml 1/2 TSB and grown in its respective temperatures until an optical density at 600 nm (OD600) of 1.0 was attained. Then 1 ml of this culture was used to inoculate 100 ml of 1/2 TSB in a Nephlo Flask (Belco) for the experiment.
|
| Growth protocol |
All the cells for the DNA microarray experiments came from the same E. sibiricum 255-15 frozen stock that was used for the genome sequencing. All experiments were performed by first plating the cells in 1/2 Tryptic Soy Agar (TSA) and then transferred to 1/2 Tryptic Soy Broth (TSB) twice. In total, six samples were grown independently at 39°C, 28°C, 10°C and -2.5°C. The cells were acclimated by growth at 39°C, 28°C, 10°C and -2.5°C. For the growth at 39°C and 28°C the plates were incubated overnight. At 10°C, the plates were incubated for 3 to 4 days. At -2.5°C the plates were transferred three times to new plates to acclimate the cells to lower temperature as follows: the first plates were incubated overnight at 22°C followed by incubation at 4°C for 3 days and then for 2-3 weeks at -2.5°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The samples were incubated until reaching mid-log growth (0.1 < OD600 < 0.3) when 100 ml of RNAlater (Ambion, Austin, Texas) was added at the same temperature as the grown cells. Cells were pelleted by centrifugation at 5,000 X g for 20 min at 4°C and resuspended in 1 ml of RNAlater, transferred to a 1.5 ml microcentrifuge tube, and re-pelleted at 5,000 X g at 4°C for 10 min.The cells were re-suspended in 100 ul of RNase-free 3 mg ml-1 Lysozyme in TE buffer pH 8 (50 mM Tris-Cl and 1 mM EDTA) by vortexing and then incubated at room temperature for at least 20 min or until the pellet cleared. The RNA was then isolated using the RNeasy mini Prep kit (Qiagen) according to the manufacturer's instructions; the step of DNase digestion was included. The resulting RNA was checked by denaturing agarose gel electrophoresis for DNA contamination and for the presence and integrity of the rRNA bands. The amount of RNA was quantified using a UV-spectrophotometer at OD260.
|
| Label |
Cy3
|
| Label protocol |
Amino-allyl labeling was performed as adapted from a protocol of The Institute for Genomic Research (TIGR) (http://www.tigr.org/tdb/microarray/protocolsTIGR.shtml). Briefly, 10 ug of total RNA was used to synthesize cDNA overnight at 42°C using 0.5 mM of Random Hexamer Primers (Invitrogen, Carlsbad, CA), 3:2 ratio of 5-(3-amino-allyl)-dUTP and dTTP (Ambion), and Superscript II reverse transcriptase (Invitrogen), and subsequently labeled by coupling reactive Cy5 or Cy3 fluorophores (Amersham, Piscataway, NJ) to the amino-allyl groups. Purification after cDNA synthesis and chemical coupling were performed using QiaQuick PCR purification columns (Qiagen) as described in TIGR protocol. The quantity of labeled cDNA and the fluorophore incorporation efficiency were determined by using UV-visible spectrophotometry.
|
| |
|
| |
| Hybridization protocol |
Microarray slides were incubated for 60 min at 46°C with prehybridization solution (50% Ultrapure formamide (Invitrogen), 5x SSC, 0.1% SDS and 0.1 mg ml-1), washed three times in double-distilled water and one time in isopropanol, and dried by centrifugation at 50 X g for 3 min. Two cDNA's from different temperatures were mixed for direct comparisons for all temperature combinations. Each microarray received about 30 ul of hybridization solution (50% Ultrapure Formamide, 5x SSC, 0.1% SDS, 0.1 ug/ul Salmon sperm DNA) containing the two cDNAs. The solution was applied by capillary action under a coverslip (LifterSlip; Erie Scientific Company, Portsmouth, NH) placed over the microarray. The whole assembly was sealed in a hybridization chamber (CMT Hybridization Chamber; Corning Incorporated, Corning, NY) and submerged for 16 h in a 46°C water bath. Microarray slides were washed twice for 5 min at 46°C with 1X SSC-0.1% SDS; twice for 10 min at room temperature with 0.1X SSC-0.1% SDS and five times for 1 min at room temperature with 0.1 X SSC. Slides were dried by centrifugation at 50 X g for 3 min and were immediately scanned and analyzed.
|
| Scan protocol |
Slides were scanned with an Axon 4000B scanner and GenePix 5.0 used for spot finding. Only spots with more than 80% of pixels greater than background plus 2 standard deviations in either Cy5 or Cy3 channel were used for analysis.
|
| Description |
Biological replicate 6 of 6 and dye swap
|
| Data processing |
Analysis was performed with Limma (Linear models for microarrays data) library in the CARMAweb environment. The background correction was done by background subtraction of the median value, followed by within and between arrays data normalization using the print tip Lowess method, and quantile method, respectively.
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|
| Submission date |
Jan 10, 2008 |
| Last update date |
Jan 10, 2008 |
| Contact name |
Debora Frigi Rodrigues |
| E-mail(s) |
[email protected]
|
| Fax |
517-353-2917
|
| Organization name |
Michigan State University
|
| Department |
Microbiology and Molecular Genetics
|
| Lab |
Center for Microbial Ecology
|
| Street address |
540 Plant and Soil Science Bldg.
|
| City |
East Lansing |
| State/province |
MI |
| ZIP/Postal code |
48824 |
| Country |
USA |
| |
|
| Platform ID |
GPL6358 |
| Series (1) |
| GSE10133 |
Architecture of thermal adaptation in Exiguobacterium sibiricum strain 255-15: A genome and transcriptome approach |
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