|
| Status |
Public on Jan 15, 2012 |
| Title |
L.monocytogenes (mono culture) in biofilm after 24 hrs of incubation |
| Sample type |
RNA |
| |
|
| Source name |
L.monocytogenes (mono culture) in biofilm after 24 hrs of incubation
|
| Organism |
Listeria monocytogenes J0161 |
| Characteristics |
growth state: Biofilm, Mono-culture
incubation hours: 24 hrs
|
| Treatment protocol |
Cells from the broth were pelleted directly. For cells from biofilm the slides immeresed in the petridish were removed of excess media, cells from the slide (biofilm) were scrapped using scalpe to microfuge tubes and pelleted there after. Cells pelleted were stored in RNA later (Ambion) till extraction.
|
| Growth protocol |
Overnight cultures were used for inoculation. For broth 1ml (each) of the overnight cultures were inoculated to freshly prepared Tryptone Soy Broth in a tube and incubated at 37 C for the required period of time intervals. For biofilm 1ml (each ) of the overnight cultures were inoculated into freshly prepared Tryptone Soy Broth in a Petridish. Glass slide was immeresed in the broth in the petridish and incubated at 37 C for the required period of time.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction was done using standard protocol of RiboPure Bacterial RNA extraction method (Ambion - AM 1925)
|
| Label |
cy3
|
| Label protocol |
Poly (A)-tails were added to the 3’-end of RNA by using A-plus Poly (A) polymerase tailing kit (Epicentre Biotechnologies).Then the samples were labeled using Agilent Quick Amp Kit PLUS (Part number: 5190-0442). Five hundred nanograms each of the samples were incubated with reverse trancription mix at 42°C and converted to double stranded cDNA primed by oligodT with a T7 polymerase promoter. The cleaned up double stranded cDNA were used as template for aRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up and quality assessed for yields and specific activity.
|
| |
|
| Hybridization protocol |
600 ng of cy3 labeled samples were fragmented and hybridized. Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of Agilent (Part Number 5188-5242). Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Part No: 5188-5327)
|
| Scan protocol |
Scanned on an Agilent Technologies G2505C scanner and Images were quantified using Agilent Feature Extraction Software 10.5.1.1
|
| Description |
Sample 3A
|
| Data processing |
The normalization was done using GeneSpring GX 11 Software.
|
| |
|
| Submission date |
Mar 14, 2011 |
| Last update date |
Jan 15, 2012 |
| Contact name |
Prem Saran Tirumalai |
| E-mail(s) |
[email protected]
|
| Phone |
+91 9410421149
|
| Organization name |
Dayalbagh Educational Institute
|
| Department |
Faculty of Science
|
| Street address |
Dayalbagh
|
| City |
Agra |
| State/province |
Uttar Pradesh |
| ZIP/Postal code |
282110 |
| Country |
India |
| |
|
| Platform ID |
GPL13262 |
| Series (1) |
| GSE27936 |
Gene Expression Profiling of Listeria monocytogenes in monoculture and in co-culture state in the presence of Bacillus subtilis both as planktonic cells and in biofilms. |
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