|
| Status |
Public on Feb 24, 2010 |
| Title |
Nicotinamide Starve of Mycobacterium bovis Defective for de novo NAD Biosysthesis Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
whole_organism from Mycobacterium bovis Ravenel Delta nadABC aka mc2 4957
|
| Organism |
Mycobacterium tuberculosis variant bovis |
| Characteristics |
phenotype: requires 0.1mg/L nicotinamide for growth
strain: Delta
|
| Biomaterial provider |
Catherine Vilcheze at HHMI at Albert Einstein CoM
|
| Treatment protocol |
nucleic_acid_extraction: total RNA prep
|
| Growth protocol |
grown with 20mg/L nicotinamide
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells are collected by centrifugation and resuspended in 1mL buffer RLT from a Qiagen RNeasy kit. The suspension is transferred to Fast-Prep Blue Cap tubes and processed for 45 seconds at speed 6.5 in a Fast-Prep apparatus. After a brief incubation on ice the debris is spun down and the supernatant (~750uL) is removed to a fresh microcentrifuge tube. 525uL absolute ethanol is added and the samples are applied to RNeasy columns in two applications. At this point the RNA is purified as recommended by the Qiagen protocol.
|
| Label |
Cy3
|
| Label protocol |
2 ug total RNA was used as template for reverse transcription with random hexamer primers and aminoallyl-dUTP in a 2:3 ratio to dTTP in the nucleotide mix. After overnight incubation at 42°C, the reaction was stopped, RNA was hydrolyzed, and unincorporated nucleotides and free amines were removed with the Qiagen MinElute kit as per protocol, with amine-free phosphate wash and elution buffers. cDNA concentration and purity were measured by Nanodrop. Cy3 and Cy5 dyes were coupled to the aminoallyl-cDNA, which was then purified using a MinElute kit. Probe concentration, purity, and dye incorporation were measured by Nanodrop.
|
| |
|
| Channel 2 |
| Source name |
whole_organism from Mycobacterium bovis Ravenel Delta nadABC aka mc2 4957
|
| Organism |
Mycobacterium tuberculosis variant bovis |
| Characteristics |
phenotype: requires 0.1mg/L nicotinamide for growth
strain: Delta
|
| Biomaterial provider |
Catherine Vilcheze at HHMI at Albert Einstein CoM
|
| Treatment protocol |
nucleic_acid_extraction: total RNA prep
|
| Growth protocol |
grown without 20mg/L nicotinamide
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells are collected by centrifugation and resuspended in 1mL buffer RLT from a Qiagen RNeasy kit. The suspension is transferred to Fast-Prep Blue Cap tubes and processed for 45 seconds at speed 6.5 in a Fast-Prep apparatus. After a brief incubation on ice the debris is spun down and the supernatant (~750uL) is removed to a fresh microcentrifuge tube. 525uL absolute ethanol is added and the samples are applied to RNeasy columns in two applications. At this point the RNA is purified as recommended by the Qiagen protocol.
|
| Label |
Cy5
|
| Label protocol |
2 ug total RNA was used as template for reverse transcription with random hexamer primers and aminoallyl-dUTP in a 2:3 ratio to dTTP in the nucleotide mix. After overnight incubation at 42°C, the reaction was stopped, RNA was hydrolyzed, and unincorporated nucleotides and free amines were removed with the Qiagen MinElute kit as per protocol, with amine-free phosphate wash and elution buffers. cDNA concentration and purity were measured by Nanodrop. Cy3 and Cy5 dyes were coupled to the aminoallyl-cDNA, which was then purified using a MinElute kit. Probe concentration, purity, and dye incorporation were measured by Nanodrop.
|
| |
|
| |
| Hybridization protocol |
Slide was hybridized for 16 hours at 42 in water bath with volume of 60 uL in solution of standard gene expression hybridization solution, and blocked in standard prehybridization solution. After hybridization, the slide was washed with standard wash protocol.
refer to PFGRC protocol SOP#M008
|
| Scan protocol |
scanned with PMT voltage adjusted for maximum dynamic range
|
| Description |
effect of nicotinamide starvation in mycobacteria defective for de novo NAD biosynthesis
|
| Data processing |
In MIDAS, lowess normalization, standard deviation regularization, in-slide replicate analysis, one bad channel policy set to generous, all flags on
|
| |
|
| Submission date |
Nov 05, 2009 |
| Last update date |
Dec 21, 2009 |
| Contact name |
Brian Weinrick |
| Organization name |
Trudeau Institute
|
| Street address |
154 Algonquin Avenue
|
| City |
Saranac Lake |
| State/province |
NY |
| ZIP/Postal code |
12983 |
| Country |
USA |
| |
|
| Platform ID |
GPL5774 |
| Series (1) |
| GSE18909 |
Nicotinamide Starvation of Mycobacteria Defective for de novo NAD Biosynthesis |
|
