3 protocols
nucleic acid hybridization to array protocol
Microarray slides were prehybridised in 3.5 x SSC, 0.1 % SDS and 10 mg/ml BSA (Sigma) for 20 minutes at 60C and then washed for one minute in water and one minute in isopropanol. Slides were then placed inside 50 ml Falcon tubes and centrifuged at 300 rcf for five minutes to dry the slides. Labelled samples were competitively hybridised against a single microarray using two Lifterslips (Thermo Scientific) to cover the array, combining the samples and then pipetting the sample under the Lifterslips allowing the solution to cover the microarray by capillary action. Microarray slides were placed in hybridisation chambers, submerged in water and incubated in a 65C oven for 16 to 24 hours. Slides were then washed vigorously for two minutes in 1 x SSC, 0.05 % SDS followed by two washes in 0.06 x SSC for two minutes each.
nucleic acid labeling protocol
5ug of total RNA was labelled with 3 ug of random primers (Invitrogen) in the presence of 500 U SuperScript II Reverse transcriptase (Invitrogen), 1x first strand buffer, 10 mM DTT, 5 mM dA/G/TTP, 2 mM dCTP and 1.5 nmol Cy3 or Cy5 labelled dUTP. RNA and random primers were heated at 95C for five minutes in a final volume of 11 ul and snap cooled for two minutes. 500 U SuperScript II Reverse transcriptase (Invitrogen), 1x first strand buffer, 10 mM DTT, 5 mM dA/G/TTP, 2 mM dCTP and 1.5 nmol Cy3 or Cy5 labelled dUTP was then added to the RNA and random primer mix and incubated at 25C for 10 minutes, followed by 42C for 90 minutes to allow cDNA synthesis to occur.
nucleic acid extraction protocol
Total RNA was isolated from 100 ml of exponential phase (OD600 0.6) rolling cultures using the Fast RNA Pro Blue kit (Qbiogene) as per the manufacturers guidelines. Cultures were harvested by centrifugation and then resuspended in 2 ml RNAPro solution. The resuspension was transferred into two matrix tubes and ribolysed (Hybaid) on setting six for 40 seconds to release the cellular RNA, DNA and proteins. The tubes were centrifuged at 13, 000 rcf for 5 minutes at 4 oC then the upper phase was transferred to a new tube. 300ul of chloroform was added, tubes were vortexed and then incubated for five minutes at room temperature before being centrifuged as previously. The upper aqueous phase containing the RNA was transferred to a new tube. RNA was precipitated by addition of 500 ul of 100 % ethanol and incubated at - 20C overnight. The RNA was then pelleted by centrifugation and washed in 70 % ethanol (made with DEPC treated water). The pellet was allowed to air-dry and then resuspended in 100ul DEPC H2O containing 40 U RNase inhibitor (Promega). Contaminating DNA was removed by DNase digestion with 2 U RNase free DNase (Promega) in 5 mM magnesium sulphate and 100 mM sodium acetate with 80 U RNase inhibitor and incubated at 37C for one hour. A further 2 U of DNase was then added and incubated for a further hour PCR. Proteins and other contaminants were removed from RNA samples using the RNA cleanup kit (RNeasy kit, Qiagen) following the manufacturers guidelines. RNA samples were suspended in a buffer containing guanidinium thiocynate and ethanol. Samples were then applied to the spin column. Contaminants were washed away with an ethanol based wash solution and RNA was eluted twice in 30ul of RNAse-free water. RNA yield was quantified using a Nanodrop ND-100 spectrophotometer, Version 3.1.2 (Labtech).200 ng of clean RNA was then run on a 2100 Bioanalyzer (Agilent Technologies) to assess integrity.