3 protocols
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 extraction protocol
50ml of GTC solution (5M Guanidium thiocyanate, 0.5%(w/v) sodium N-lauroyl sarcosine, 25mM tri-sodium citrate, 0.1M 2-mercaptoethanol and 0.5%(w/v) Tween80 in RNAse free water (Sigma-Aldrich,Poole, UK)) was added to 50ml of culture and centrifuged. After centrifugation, the cells were resuspended in Trizol and were disrupted for 45sec (setting of 6.5) using a ribolyser (Hybaid). RNA was extracted with chloroform and precipitated with isopropanol and 0.3M sodium acetate. The precipitated RNA was spun down and washed with in 70 per cent ethanol. The pellet was air dried and dissolved in 100ul of water. A Qiagen mini RNeasy clean up kit (Qiagen, Crawley, UK) was used to purify the RNA following the manufacturers guidelines. The RNA was eluted into 30-50ul of water. Contaminating DNA was removed from the RNA preparation using the Turbo DNAfree kit (Ambion). The integrity of the RNA was checked by agarose gel electrophoresis and quantified using a spectrophotometer. Each RNA preparation yielded 5-30ug.