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| Status |
Public on Jun 01, 2013 |
| Title |
015_2h_2 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
X514 (wt) was collected at mid-log after addition 0.15% ethanol 2 hour
|
| Organism |
Thermoanaerobacter sp. X514 |
| Characteristics |
strain: Thermoanaerobacter sp. X514
treatment: 0.15% ethanol treated 2 hour
|
| Growth protocol |
Thermoanaerobacter sp. X514 wt, Xm and Xe were grown anaerobically in defined medium supplement either with or without ethanol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was isolated using Trizol Reagent (Invitrogen). RNA samples were treated with Rnase-free DNaseI and purifed using the total RNA Isolation Kit (NucleoSpin RNAII, Macherey-Nagel Inc. Genomic DNA was isolated and purified from Thermoanaerobacter sp. X514 by CTAB (hexadecyltrimethylammonium bromide)-NaCl method (Zhou et al., 1995).
|
| Label |
Cy5
|
| Label protocol |
Ten microgram of total RNA was reverse-transcribed using random primers (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen) in the presence of 1 mM Cy5 dUTP (Invitrogen) according to previous description (He et al., 2006). The purified 500 ng genomic DNA was fluorescently labeled by random priming using Klenow fragment of DNA polymerase in the presence of 1mM Cy3 dUTP as described elsewhere (He et al., 2005).
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| |
|
| Channel 2 |
| Source name |
pool of genomic DNA extracted from control organisms for all samples
|
| Organism |
Thermoanaerobacter sp. X514 |
| Characteristics |
strain: Thermoanaerobacter sp. X514
treatment: control
|
| Growth protocol |
Thermoanaerobacter sp. X514 wt, Xm and Xe were grown anaerobically in defined medium supplement either with or without ethanol.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total cellular RNA was isolated using Trizol Reagent (Invitrogen). RNA samples were treated with Rnase-free DNaseI and purifed using the total RNA Isolation Kit (NucleoSpin RNAII, Macherey-Nagel Inc. Genomic DNA was isolated and purified from Thermoanaerobacter sp. X514 by CTAB (hexadecyltrimethylammonium bromide)-NaCl method (Zhou et al., 1995).
|
| Label |
Cy3
|
| Label protocol |
Ten microgram of total RNA was reverse-transcribed using random primers (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen) in the presence of 1 mM Cy5 dUTP (Invitrogen) according to previous description (He et al., 2006). The purified 500 ng genomic DNA was fluorescently labeled by random priming using Klenow fragment of DNA polymerase in the presence of 1mM Cy3 dUTP as described elsewhere (He et al., 2005).
|
| |
|
| |
| Hybridization protocol |
The Cy3 labeled genomic DNA was used as a common reference to co-hybridize the Cy5 labeled RNA samples on each slide. Hybridization was carried out using a TECAN HS4800 Pro Hybridization Station (Tecan, NC) by following the manufacturer’s instructions. This system automatically performs hybridization, washing and drying of arrays.
|
| Scan protocol |
slides were scanned using a ProScanArrayTM microarray analysis system (Perkin Elmer®, MA).
|
| Description |
Each oligonucleotide probe had two replicates on a single slide. Additionally, six different concentrations (11, 22, 45, 90, 180 and 360 ng/ul) of genomic DNA were spotted (eight duplicates for each of the six concentrations on a single slide) as positive controls.
|
| Data processing |
To determine signal intensity of fluorescence for each spot, the 16-bit TIFF scanned images were analyzed by the software ImaGene 6.1 (Biodiscovery Inc., EI Segundo, CA). Microarray data were analyzed as previously described by Mukhopadhyay et al. (Mukhopadhyay et al., 2006; He et al., 2009b). Briefly, it includes the following major steps. (i) Empty spots with SNR <2.0 (SNR, signal-to-noise ratio), and bad spots were flagged and removed before normalization. (ii) The net signal of each spot was calculated by subtracting the background signal and adding a pseudo signal of 100 to obtain a positive value. For resulting net signals of <50, a value of 50 was used. (iii) The total signal intensity of all microarray (slides) in this experiment was normalized with the genomic DNA signal (Cy3 signal), and a normalization factor was calculated for each slide. (iv) Both Cy3 and Cy5 signal intensity of each spot were adjusted by multiplying the normalization factor calculated above. Therefore, the resultant Cy5 signal of each probe presents the normalized signal intensity for each gene. (v) A ratio R of Cy5/Cy3 signal was calculated for each spot on each array. (vi) To compare genomic analysis, the control and treatment conditions were defined respectively so as to identify the control (Rc) and treatment (Rt) ratios. And then log2 R value was calculated for each gene. (vii) Finally, to access the significant change between treatment and control, a Z score was calculated via following equation: , where 0.25 is a pseudovariance term. Typically, a cutoff |Log2R|≥1.0 and |Z|≥ 2.0 was used to determine changes with significance.
The column headers for the raw data are (separated by ;): id;probe_ID;gene_ID;pos;flag;signal_mean;bg_mean;net_sig;bg_stdev;SNR;SBR;demo;sig_stdev;meta_row;meta_col;sub_row;sub_col
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| |
|
| Submission date |
Oct 05, 2011 |
| Last update date |
Jun 01, 2013 |
| Contact name |
Lu Lin |
| E-mail(s) |
linlu623@yahoo.com.cn
|
| Organization name |
QIBEBT
|
| Street address |
189 Songling RD
|
| City |
Qingdao |
| State/province |
Shandong |
| ZIP/Postal code |
266101 |
| Country |
China |
| |
|
| Platform ID |
GPL11001 |
| Series (1) |
| GSE32630 |
the transcript profiles of Thermoanaerobacter sp. X514 (wild type) and its ethanol tolerant mutants (Xm and Xe) with or without ethanol |
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