the method recommended by BuG@S to extract data from scanned images using ImaGene
nucleic acid hybridization to array protocol
Excess Cy3 and Cy5 dCTP was removed from labelled DNA or cDNA samples using the MinElute Reaction Cleanup Kit (Qiagen). Cy3 and Cy5 labelled DNA and/or cDNA samples were combined in a single tube (1.5mL) and 5 volumes of PB buffer added. The solution was applied to a MinElute column in a collection tube and centrifuged at 13,000rpm for 1min. The flow-through was discarded and the MinElute column placed back into the same collection tube. 500uL of Buffer PE was added to MinElute column, centrifuged at 13,000rpm for 1min and the flow-through discarded. The MinElute column was placed back in the same collection tube and the previous step repeated with 250uL of PE. The MinElute column was placed into a fresh 1.5mL tube, 15.9uL of water (22x22mm LifterSlip) or 30.2uL (22x50mm LifterSlip) was added to the centre of the membrane, allowed to stand for 1min and then centrifuged at 13,000rpm for 1min. 50mL of prehybridization solution (3.5xSSC, 0.1% SDS, 10mg/mL BSA) was placed in Coplin jar and incubated at 65C to preheat for 1h 30min. The microarray slide was placed in the pre-hybridization solution and incubated at 65C for 20min. The slide was then rinsed in 400mL water for 1min and 400mL of propan-2-ol for 1min. The slide was placed in a 50mL centrifuge tube and centrifuged at 1,500rpm for 5min to dry. Each slide was stored in a dark, dust free box until hybridization (<1h). The prehybridized microarray slide was placed in the hybridization cassette and two 15uL aliquots of water added to the wells of the cassette. A 4xSSC 0.3% SDS hybridization solution containing the Cy3/Cy5 labelled samples was prepared with a final volume of 23ul (22x22mm LifterSlips)or 45uL (22x50mm LifterSlips). The hybridization solution was heated at 95C for 2min, allowed to cool slightly at room temperature and briefly centrifuged. A LifterSlip was placed carefully over the arrayed area of the slide, to avoid scratching its surface. The hybridization solution was pipetted under one corner of the LifterSlip, allowing the solution to be drawn completely across the array by capillary action. Any excess hybridization solution was pipetted under the opposite corner of the LifterSlip. The hybridization cassette was sealed and submerged in a water bath at 65C in the dark for 16-20h. Wash A (1xSSC, 0.05% SDS) was preheated to 65C and placed in a staining trough pre-heated to 65C. The microarray slide was removed from the hybridization cassette and carefully washed in the staining trough of Wash A at 65C to remove LifterSlip. The slide was then placed in a slide rack and agitated in Wash A for a further 2min. Slides were transferred to a clean rack and agitated in a trough of 400mL of Wash B (0.06xSSC) for 2min at room temperature. Slides were transferred to a second trough of 400mL of Wash B (0.06xSSC) and agitated for a further 2min at room temperature. The slide was then dried by centrifugation at 1,500rpm for 5min in a 50mL centrifuge tube.
nucleic acid labeling protocol
2-10ug of RNA sample was placed in a microfuge tube (0.5mL) with 3ug of random primers (1uL) and made up to a final volume of 11uL with H2O (DNase and RNase free, molecular biology grade). The RNA was then heated to 95C for 5min, snap cooled on ice and centrifuged. 5uL First Strand Buffer (5x), 2.5uL DTT (100mM), 2.3ul dNTP's (5mM dA/G/TTP, 2mM dCTP), 1.7uL of Cy5(1mM) and 2.5uL of SuperScript II (200U/uL) were added to make a final volume of 25uL. The solution was incubated in the dark at 25C (room temperature) for 10min and then at 42C in the dark for 90min.
nucleic acid labeling protocol
For RNA samples labelled with Cy3 dCTP. 2-10ug of RNA sample was placed in a microfuge tube (0.5mL) with 3ug of random primers (1uL)and made up to a final volume of 11uL with water (DNase and RNase free, molecular biology grade). The RNA was then heated to 95C for 5min, snap cooled on ice and centrifuged. 5uL First Strand Buffer (5x), 2.5uL DTT (100mM), 2.3ul dNTP's (5mM dA/G/TTP, 2mM dCTP), 1.7uL of Cy3(1mM) and 2.5uL of SuperScript II (200U/uL) were added to make a final volume of 25uL. The solution was incubated in the dark at 25C (room temperature) for 10min and then at 42C in the dark for 90min.
BAB-Haemin plates were inoculated with Yersinia pestis strain GB or the deletion mutant and incubated for 48h at 28C. 20mL of BAB broth were inoculated with 2-3 colonies of either the Yersinia pestis GB or the mutant strain. The cultures were incubated statically at 28C overnight. 18mL of pre-warmed BAB broth was seeded with 2mL of Y.pestis GB overnight culture. 18 or 16mL of pre-warmed BAB broth were seeded with 2 or 4mL of Y.pestis mutant overnight culture, respectively. The cultures were incubated at 28C with aeration (200rpm) until the culture reached an optical density, OD600nm, of 0.2. The cultures were then incubated at 37C and grown with aeration until they reached an OD600 of 0.4.
2mL of Yersinia pestis culture at OD600 of 0.4 was added to 4mL of RNA protect. The mixture was vortexed, incubated at room temperature for 5min and centrifuged at 7000rpm for 15min. The supernatant was removed and the pellet left to airdry for 30min. The RNA was extracted using QIAgen RNeasy extraction kit. The pellet was resuspended in 200ul TE buffer containing 1mg/mL lysozyme and incubated at room temperature for 10min, with vortexing every 2min. The manufacturer?s protocol was followed from this point, including the ?on-column? DNase treatment. RNase-free water was added to the membrane and the spin column was centrifuged after 1min at 10,000g for 1min. The concentration and purity of the total RNA was analysed using The yield was approximately 150ng/ul.
A Lowess curve was fit to the log-intensity versus log-ratio plot. 40.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 0.001 then 0.001 was used instead.