MICROARRAY HYBRIDISATION PROTOCOL 10/05 (for both PCR and Oligo arrays on UltraGAPS slides) Vassilis Mersinias, Giselda Bucca and Graham Hotchkiss Quick Protocol (see notes at end for detailed instructions) Use the following Cy-dye labelled dctp volumes for the appropriate application Application Vol. Cy3-dCTP Vol. Cy5-dCTP cdna vs cdna 1.5µl 2.5µl cdna vs genomic 1.5µl (cdna) 1.5µl (gdna) genomic vs genomic 1.5µl 1.5µl NB for cdna vs genomic use Cy3 for cdna labelling and Cy5 for genomic cdna synthesis and labelling RNA must be purified and dissolved in H 2 O see notes and purification protocol Optional: If you wish to make use of the Lucidea Universal ScoreCard control spots add 2µl of reference and test mrna spike mixes to the respective bacterial RNA samples (for cdna vs cdna hybridisations). See notes at end for ordering the mrna spike mixes. Read supplier s manual for details. For each sample: 10-15µg total RNA (15.2µl max for Cy3 labelling 14.2µl max for Cy5) 1.7µl Random primers to 16.9µl (Cy3) OR 15.9µl (Cy5) RNase-free H 2 O 70 o C for 10min, snap cool on ice, spin down. Then add (make a master mix for multiple labellings): 6µl 5x First StrandBuffer 3µl 100mM DTT 0.6µl dntps (25mM each da/g/ttp, 10mM dctp) 2µl SuperScript II or III 1.5µl Cy3-dCTP OR 2.5µl Cy5-dCTP 25 o C for 10 min in the dark. 42 o C for 110min in the dark. UniS Streptomyces microarray group. Hybridisation protocol October 05, page 1 of 6
Denature RNA To each RNA labelling reaction: Add 10µl 1N NaOH 70 o C for 10min Add 10µl 1N HCl Genomic DNA labelling Labelling For each reaction: 2-3µg genomic DNA (sonication not necessary) 1µl Random primers to 41.5µl ddh 2 O 95 o C for 5min, snap cool on ice and briefly centrifuge. Then add: 5µl Klenow Buffer (10x) 1µl dntps (5mM each da/g/ttp, 2mM dctp) 1.5µl Cy3-dCTP OR Cy5-dCTP 1µl Klenow fragment (5U/µl) 37 o C for 90min to overnight in the dark (see notes). Pre-hybridisation For pre-soaking and pre-hybridisation of the slides use the Pronto! Universal Microarray Hybridisation kit (Corning, Cat.# 40026) according to the supplier s instructions. Purification using Qiagen MinElute columns To each labelling reaction: Add 250µl (5 volumes) buffer PB and spin through column 1min at 13K Add 500µl buffer PE, spin 1min at 13K Add 250µl buffer PE, spin 2min at 13K Transfer column to clean tube Add 10µl buffer EB (or MilliQ H 2 O ph 7-8.5), 1min at RT, spin 1min at 13K Add another 10µl buffer EB (or MilliQ H 2 O ph 7-8.5), 1min at RT, spin 1min at 13K For each Cy-cDNA sample measure the concentration of Cy nucleotides spectrophotometrically at 550nm (Cy3) or 650nm (Cy5): UniS Streptomyces microarray group. Hybridisation protocol October 05, page 2 of 6
pmol Cy3/µl = A 550 / 0.15 pmol Cy5/µl = A 650 / 0.25 Take into account any dilutions of the original eluate prior to spectrophotometry. We recommend the use of Nanodrop spectrophotometer where readings are provided directly from just 1µl of undiluted sample. Take an aliquot of Cy-cDNA containing 45pmol of Cy nucleotides and/or an aliquot of Cy-gDNA containing 10-20 pmol of Cy-dCTP (see Notes: Labelling efficiency). Mix Cy3 and Cy5 samples together and dry the labelled targets completely in a SpeedVac at RT. Hybridisation Use 45µl of Pronto! cdna/longoligo Hybridisation Solution* (supplied with the Pronto! Universal Microarray Hybridisation kit (Corning, Cat.# 40026)) to redissolve the labelled probes (final concentration of each cdna-incorporated Cy dye 1pmol/µl). *Do not use the Pronto! Short Oligo Hybridisation Solution provided in the same kit (this applies to both PCR and Oligo arrays). Denature at 95 o C for 5min Pre-heat pre-hybridised slide and place 30µl 3x SSC in each well of the hybridisation chamber towards end of incubation. Spin down probe and immediately transfer to arrayed area of slide, avoiding bubbles. Quickly place 24 x 60mm cover slip over and seal in hybridisation chamber. Incubate in 42 o C water bath for 16-20 hrs. Washing For post-hybridisation washes of the slides use the Pronto! Universal Microarray Hybridisation kit (Corning, Cat.# 40026) according to the supplier s instructions. Dry by centrifugation or compressed air UniS Streptomyces microarray group. Hybridisation protocol October 05, page 3 of 6
Notes General precautions and tips Minimise the exposure of Cy dyes to the light. Use RNase-free pipette tips, tubes and H 2 O for RNA labelling reaction (e.g. RNasefree 1.5ml tubes and non-depc treated H 2 O from Ambion, ART filtered tips from Fisher). RNA preps should be at a concentration of at least 0.7µg/µl and must be purified to remove excess salts that may interfere with the labelling reaction. Due to better signal-to-noise ratios for cdna in the Cy3 channel we recommend the labelling of cdna with Cy3 and of genomic DNA with Cy5. However if you wish to do a dye swap use 2.5µl Cy5-dCTP for cdna and 1.5µl Cy3-dCTP for genomic DNA. Genomic DNA should be from a stationary phase culture, otherwise genes nearer the origin of replication will be over represented. Labelling efficiency You can incubate the cdna labelling reaction at 42 o C for longer than 2 hrs (4-5 hrs) for higher yield. Expect a Cy dye concentration in the pure Cy-cDNA of ca. 10 pmol/µl or more for a successful reaction. To save reagents and RNA you can scale down the Cy-cDNA synthesis reaction to half of the amount of RNA and half of the volume of each reagent. SuperScript III may give higher yields of labelled cdna than SuperScript II, but it is more expensive. We do not find the use of SuperScript III essential for a successful reaction. If the purified cdna eluate is virtually colourless do not proceed, your RNA did not yield labelled cdna. Check the quality of the RNA and/or the other reaction components and store the labelled gdna at 20 o C for use in another hybridisation. It is completely normal for purified Cy-labelled genomic DNA to have a faint colour. If, however, the gdna doesn t label (colourless eluate and less than 1 pmol/µl of incorporated Cy-dCTP) you can try to further purify your DNA preparation through GFX columns (GE Healthcare, GFX PCR and Gel Band Purification Kit cat no. 27-9602-01) and incubate the reaction for longer (it is OK to run the reaction overnight). While we use 40pmol of Cy-cDNA for array hybridisation according to Corning s recommendations, Cy-gDNA needs substantially less amount: 10-20 pmol of labelled gdna is sufficient for successful hybridisation. When using gdna as common reference on multiple arrays (e.g. in a time course experiment) you can set a multiple reaction a single tube and split the purified labelled gdna so that 10-20 pmol of Cy-gDNA is added per array. To use the same amount of Cy-gDNA from the same reaction on all the arrays of the experimental set is probably more important than exactly how much Cy-gDNA to use (10 or 15 or 20 pmol). UniS Streptomyces microarray group. Hybridisation protocol October 05, page 4 of 6
Hybridisation tips and tricks Once hybridisation solution has been added to the probes keep it at room temperature to avoid precipitation of SDS. Transfer of the probe to the slide must be done carefully but quickly to prevent renaturation of probes and extended evaporation of the sample. Prepare the slide and cover slip by removing any dust with pressurised air and pre-heat it on a slide heating block (set to 7 / about 50 0 C) or standard heat block with the wells face down. Position the cover slip ready to pick up. Apply the probe as a line in the middle of the spotted area avoiding touching the slide surface and bubble formation. This is best achieved by not expelling the final drop of liquid. Using curved-edge forceps place one edge of the cover slip just outside the spotted area at an angle to the slide surface. Hold a microspatula down on the slide surface close to (or just touching) the cover slip to prevent it from sliding away from the arrayed area. Carefully lower the other edge of the cover slip down with the forceps and lay it down on the probe, which should spread out under the cover slip. Once in place do not move the cover slip (unless it is misplaced accidentally). Small bubbles usually migrate out of the cover slip. Check after a few minutes in the water bath and if necessary gently tap the cover slip to dislodge any stubborn ones. Wash the microspatula and forceps in between slides if they come into contact with the probe. Washing and drying slides Use slide staining troughs of 100ml capacity (optimum volume for use with Pronto! kit solutions). Do not touch the arrayed area, even with gloves Wash by incubation or gentle agitation on an orbital shaker (see instructions in Pronto! manual) Minimise exposure of the slide to the air during washing SDS is fluorescent - be particularly careful of what is on your gloves A slide staining rack (minus the handle) sandwiched between two microtitre plate lids, with a little absorbent cloth to cushion the slides and catch liquid, makes an excellent device to spin dry the slides. Spin in a centrifuge rotor that takes microtitre plates at 800rpm for 5min. Balance using an identical structure with the same number a slides. Alternatively spin in 50ml tubes at 800rpm for 5min with arrayed side of slides facing up. UniS Streptomyces microarray group. Hybridisation protocol October 05, page 5 of 6
Required equipment / consumables Scanner and image analysis software Orbital shaker Centrifuge with rotor for microtitre plates or 50ml tubes Microfuge Water baths / heat block / thermocycler Microscope slide staining troughs (Fisher Cat.# MNK-830-A) MinElute PCR purification kit (Qiagen Cat.# 28004) Pronto! Universal Microarray Hybridisation kit (Corning, Cat.# 40026) Slide heating block (Scientific Laboratory Supplies Cat. # MIC 4302) or heat block Hybridisation chambers (CMT -Hybridization Chambers (5), Corning Cat. # 2551) Air-duster (Dust-Pro Pressurized Duster, Sigma Cat # Z37,952-2) 24 x 60 mm cover slips (HybriSlips, Sigma Cat.# H-0784) SuperScript II (200U/µl; Invitrogen Cat.# 18064-014) Klenow fragment dntps Cy3-dCTP (Amersham Biosciences Cat.# PA53021) Cy5-dCTP (Amersham Biosciences Cat.# PA55021) Random primers (3µg/µl; Invitrogen Cat.# 48190-011) Optional: Universal ScoreCard Refill (mrna spike mixes) (GE Healthcare, formerly Amersham Biosciences, Cat.# RPK 3164) http://www.surrey.ac.uk/sbms/fgenomics/ microarrays@surrey.ac.uk UniS Streptomyces microarray group. Hybridisation protocol October 05, page 6 of 6