|
| Status |
Public on Jan 29, 2009 |
| Title |
Clostridium_acetobutylicum_rDNA_butyrate_stress_0.6%_0min |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
C. acetobutylicum (pSOS95del) plasmid control not stressed 0 min
|
| Organism |
Clostridium acetobutylicum |
| Characteristics |
Clostridium acetobutylicum ATCC 824 (pSOS95del)
|
| Biomaterial provider |
Originally purchased from American Type Culture Collection (Manassas, VA, USA).
|
| Treatment protocol |
This culture is the control strain for this experiment. The culture was challenged with 0.6% butyrate at an OD of 1.0 (mid-exponential growth). Before challenging, the butyrate was adjusted to pH 6.7 (the approximate pH of the growing culture). This sample was taken immediately prior to addition of butyrate.
|
| Growth protocol |
Colonies of C. acetobutylicum ATCC 824 (pSOS95del) (American Type Culture Collection, Manassas, VA, USA) were grown on agar-solidifed 2xYTG plates (YTG is 8 g/liter tryptone, 5 g/liter yeast extract, 2 g/liter NaCl, 2.5 g/liter glucose, and 7.5 g/l agar, pH 5.8) containing 40 micrograms/mL of erythromycin. Liquid cultures were inoculated with single colonies from plates at least 4 days old and heat shocked at 70 to 80°C for 10 min. Cultures were grown in an anaerobic chamber (Forma Scientific, Marietta, OH) in Clostridial Growth Medium (CGM, Wiesenborn, D. P., et al. 1988. Appl. Environ. Microbiol. 54:2717–2722) supplemented with 80 g/liter glucose and 100 micrograms/mL erythromycin until they reached an A600 ca. 1.0 (corresponding to midexponential growth). Butyric acid, adjusted to pH 6.7 with 10M sodium hydroxide, was then added to 0.6%v/v final concentration.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were collected by centrifuging 3 to 10 mL of cultures at 5,000xg for 10 min, 4 degrees C. Cells were then lysed by resuspending in 200 microL SET buffer (25% sucrose, 50 mM EDTA [pH 8.0], 50 mM Tris [pH 8.0]) with 20 mg/mL lysozyme (Sigma) and incubation at 37 degrees C for 4 min. One milliliter of ice cold TRIzol (Invitrogen, Carlsbad, CA, USA) was added and the sample was vortexed. Samples were immediately stored at -85 degrees C and used within one month to minimize RNA degradation. To isolate and purify the RNA, samples were thawed at room temperature; 500 microL of sample was diluted with an equal amount of ice cold TRIzol and then purified following manufacturer’s instructions. The RNA was resuspended in 20 microL RNase-free water and quantitated in a spectrophotometer by measuring absorbance at 260 and 280 nm. Samples were only used if the A260/A280 ratio was greater than 1.8. RNA integrity was also verified by running 0.5 microL of sample on a 1.0% agarose gel. Purified RNA were stored at -85 degree C and used within one week of purification.
|
| Label |
Cy5
|
| Label protocol |
An indirect labeling approach was followed where cDNA was first made in the presence of nucleotides and aminoallyl dUTP, purified, and then the aminoallyl dUTP groups were coupled to a monoreactive dye (Cy3 or Cy5) as described previously (Alsaker et al. 2005. Biotechnology and Bioprocess Engineering 10: 432-443).
|
| |
|
| Channel 2 |
| Source name |
C. acetobutylicum (RDNA7) not stressed 0 min
|
| Organism |
Clostridium acetobutylicum |
| Characteristics |
Clostridium acetobutylicum ATCC 824 (pRDNA7)
|
| Biomaterial provider |
Originally purchased from American Type Culture Collection (Manassas, VA, USA).
|
| Treatment protocol |
This culture is the experimental strain. The culture was challenged with 0.6% butyrate at an OD of 1.0 (mid-exponential growth). Before challenging, the butyrate was adjusted to pH 6.7 (the approximate pH of the growing culture). This sample was taken immediately prior to addition of butyrate.
|
| Growth protocol |
Same as channel 1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Same as channel 1
|
| Label |
Cy3
|
| Label protocol |
Same as channel 1
|
| |
|
| |
| Hybridization protocol |
Arrays were hybridized according to Agilent’s “Hybridization Procedure for 22k microarrays (cDNA labeled Targets)” (Agilent 60-mer Oligo Microarray Processing Protocol v. 4.1, Agilent); however, the 17 hr incubation was performed at 65 degrees C. Washing of the arrays was performed according to Agilent’s “Wash Procedure with Stabilization and Drying Solution” (Two-Color Microarray-Based Gene Expression Analysis v. 4.0.1, Agilent). Removal of microarrays from the final Stabilization and Drying Solution wash was immediately repeated for slides that were not completely dry.
|
| Scan protocol |
Scanned using a G2565BA Agilent Microarray Scanner (Agilent, Wilmington, DE, USA) at 10 micrometers resolution using Agilent’s eXtended Dynamic Range technique. Each array was scanned twice: once at photomultiplier tube gain of 100%, and again at a 10% gain.
|
| Description |
No additional information.
|
| Data processing |
Microarrays were scanned using a G2565BA Agilent Microarray Scanner (Agilent) at 10 microm resolution. Scans were performed using Agilent’s eXtended Dynamic Range technique. Each array was scanned twice: once at photomultiplier tube gain of 100%, and again at a 10% gain. Information from both scans was processed by Agilent Feature Extraction software (v. 9.1), used to assess spot quality and obtain feature intensity statistics; duplicate spots were averaged. Background-subtracted data were normalized using a segmental nearest neighbor logarithmic expression ratio-of-the mean (SNN-LERM) approach (Yang et al., PNAS 2003; 100:1122-1127).
|
| |
|
| Submission date |
Apr 03, 2007 |
| Last update date |
Jan 28, 2009 |
| Contact name |
Eleftherios Terry Papoutsakis |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Delaware
|
| Department |
Chemical Engineering
|
| Street address |
15 Innovation Way
|
| City |
Newark |
| State/province |
DE |
| ZIP/Postal code |
19711 |
| Country |
USA |
| |
|
| Platform ID |
GPL4412 |
| Series (1) |
| GSE14433 |
Clostridium acetobutylicum ATCC 824 (pRDNA7) butyrate stress (0.6%) |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
Normalized natural log (Sample/Control). |
| CH1_MEDIAN_INT |
Channel 1 median intensity |
| CH1_MEDIAN_BKG |
Channel 1 median background intensity |
| CH1_BKG_STD_DEV |
Channel 1 background intensity standard deviation |
| CH2_MEDIAN_INT |
Channel 2 median intensity |
| CH2_MEDIAN_BKG |
Channel 2 median background intensity |
| CH2_BKG_STD_DEV |
Channel 2 background intensity standard deviation |
| CONF_DIFF_EXPR |
Confidence of differential expression (per method of Yang et al., PNAS 2003; 100:1122-1127 [0,1] |
