|
| Status |
Public on Jun 23, 2016 |
| Title |
Biofilm versus planktonic cells replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Biofilm cells
|
| Organism |
Leptospirillum ferrooxidans |
| Characteristics |
sample: Environmental sample
strain: RT32a
|
| Treatment protocol |
Cells from water samples were collected by filtration and maintained in RNAlater. Filament samples growing in the same site were collected in RNA later. All samples were frozen on dry ice and kept at -20ºC until use.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following manufacturer's instructions. RNA samples were amplified as described in Moreno-Paz, M and Parro V.(Environ. Microbiol 2006, 8:1064-1073).
|
| Label |
Cy5
|
| Label protocol |
Labeled cDNA synthesis was achieved from 2.5ug of amplified RNA with the CyScribe first-strand cDNA labeling kit. (Amersham) and purified with GFX columns (Amersham).
|
| |
|
| Channel 2 |
| Source name |
Planktonic cells
|
| Organism |
Leptospirillum ferrooxidans |
| Characteristics |
sample: Environmental sample
strain: RT32a
|
| Treatment protocol |
Cells from water samples were collected by filtration and maintained in RNAlater. Filament samples growing in the same site were collected in RNA later. All samples were frozen on dry ice and kept at -20ºC until use.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following manufacturer's instructions. RNA samples were amplified as described in Moreno-Paz, M and Parro V.(Environ. Microbiol 2006, 8:1064-1073).
|
| Label |
cy3
|
| Label protocol |
Labeled cDNA synthesis was achieved from 2.5ug of amplified RNA with the CyScribe first-strand cDNA labeling kit. (Amersham) and purified with GFX columns (Amersham).
|
| |
|
| |
| Hybridization protocol |
Microarrays were blocked by incubation in prehybridization buffer: 5XSSC (3 M sodium chloride and 300 mM trisodium citrate (adjusted to pH 7.0 with HCl)), 0.1% SDS, 0.1ug/ul of shared denatured salmon sperm DNA and 1% BSA) at 42ºC for 45min. Hybridizations were performed overnight, at 55ºC, with 1X HybIt® Hybridization Solution (Arrayit corporation) in a volume of 50ul. 22 X 60mm coverslips (Sigma) and an ArrayIt® Hybridization Cassette (AHC, Arrayit corporation) were used. Three washing steps were carried out for 5 min in the following buffers: 1) 2xSSC + SDS 0.1%; 2) 0.2x SSC + SDS 0.1%; 3) 0.2x SSC.
|
| Scan protocol |
Images were quantified using Genepix pro 6.0 Software in a Genepix personal 4100A scanner
|
| Description |
Samples were collected from a permanent spring coming out under a pile of pyrite-containing rocks accumulated by mining activities.
collection: Environmental samples were collected from a permanent spring coming out under a pile of pyrite-containing rocks accumulated by mining activity.
|
| Data processing |
The local background was subtracted and the Log2 of processed Red signal/processed Green signal was calculated with Genepix pro 6.0 software (Molecular Devices). LOWESS normalization and statistical student's t-test analyses were carried out using AlmaZen software (BioAlma).
|
| |
|
| Submission date |
Feb 10, 2010 |
| Last update date |
Jun 23, 2016 |
| Contact name |
Mercedes Moreno |
| Organization name |
Centro de Astrobiología
|
| Street address |
Crta. de Ajalvir, km 4
|
| City |
Torrejón de Ardoz |
| State/province |
Madrid |
| ZIP/Postal code |
28850 |
| Country |
Spain |
| |
|
| Platform ID |
GPL10022 |
| Series (1) |
| GSE20267 |
Transcriptome analysis of LfeRT32a in its natural microbial community comparing the biofilm and planktonic modes of life |
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