1. Turn power on scanner and restart the PC (so the PC can recognise the scanner) ¡V Note that the lasers need 15 min to warm up before use. 2. Open GenePix Pro analysis program. 3. Open the sliding door of scanner and insert microarray into the holding clamp (DNA side facing down!). 4. Open the Hardware Settings box and set the PMTs to 600, Laser power to 100%, Focal position to 0, Pixel size to 10 ƒÝm, and Lines to average to 2. 5. Start a low resolution scan by clicking the Preview Scan button ¡V this will display a low resolution image of your microarray. 6. Define area of interest using the Scan Area button and dragging over the area with spots. 7. During low resolution scans adjust the PMTs in the Hardware Settings box so that the two channels (Cy3=532nm=green channel, Cy5=635nm=red channel) are balanced. This is carried out by moving between the Image tab and the Histogram tab at top of display. The image should have only a few pixels ¡¥off the scale¡¦ (turning white), and the histogram should show the two wavelengths having equal distribution. This may take more than one low resolution but try and minimise the number of scans to reduce photo bleaching of Cy dyes. 8. Once the image is balanced carry out a high resolution scan by clicking the Data Scan button. This may take several minutes. 9. Save the complete data scan as multiple image .tiff files by using the Open/Save function. (Parameters: Scanning hardware = GenePix Personal 4100A [Axon Instruments], Scanning software = GenePix Pro [Axon Instruments])
Template-switching cDNA amplification and labelling protocol For more details on validation of amplification see: Global Amplification of mRNA by Template-Switching PCR: Linearity and Application to Microarray Analysis. L. Petalidis, S. Bhattacharyya, G.A. Morris, V.P. Collins, T.C. Freeman and P.A. Lyons. Nucleic Acids Research, 31; 1-7 (2003). Required Reagents Use HPLC-purified primers, their sequences are as follows; 3’ SMART CDS primer IIA (5’-AAGCAGTGGTATCAACGCAGAGTAC-T30VN-3’) SMART IIA Oligonucleotide (5’-AAGCAGTGGTATCAACGCAGAGTACGC)r(GGG)-3’) 5’ PCR Primer IIA (5’-AAGCAGTGGTATCAACGCAGAGT-3’) Use PowerScript RT (Clontech #639501) and PCR Advantage 2 polymerase (Clontech #639021) · 0.2 ml thin wall tubes (Clontech #12225) · BioPrime DNA labelling system (Invitrogen #18094-011) · 10x low-C dNTP mix (5mM dGTP, dTTP, dATP, 2mM dCTP) (Sigma #DNTP-100A) · Auto-seq G-50 column (Amersham #27-5340-02) · Cy3-dCTP and Cy5-dCTP (Amersham #PA53023 and PA55023, respectively) · Ethanol · 3M Sodium Acetate · Nuclease-free water (Ambion) 1st Strand cDNA Synthesis 1. Preheat Tetrad to 72°C. 2. For each sample, combine the following reagents in a sterile, thin wall 0.2 ml tube: 1Ul total RNAa (250ng/Ul stock) 1Ul 3’ SMART CDS Primer IIA (10 UM) 1Ul SMART IIA Oligonucleotide (10 UM) kept in 70º 1Ul Stratagene Alien Spike RNA 1Ul dH2O 5Ul Total volume 3. Mix contents and spin the tube briefly in a micro centrifuge. 4. Incubate at 72°C for 2 min (in Tetrad, use heated lid). 5. Cool the tube on ice for 2 min. 6. Spin tube briefly to collect contents. 7. Add the following to each tube: 2Ul 5x First-strand buffer 1Ul DTT (0.1 M) 1Ul dNTP (10 mM) 1Ul Superscript III RT 8. Mix by gentle pipetting and spin tubes briefly in microphage. 9. Incubate the tubes at 42°C for 1 hr (in Tetrad, use heated lid). 10. Place tubes on ice to terminate first-strand synthesis. Notes a PCR may be optimised to given sample type and array. Amplifications and hybridisations using only 50 ng of cell line total RNA have with as little as 13 PCR cycles have produced acceptable results. cDNA Amplification by LD PCR 1. Transfer a 1Ul aliquot from the first-strand reaction to a clean, pre-chilled 0.2 ml tube, place on ice. Store any unused first-strand reaction mixture at –20oC. 2. Preheat Tetrad to 95°C. 3. Prepare a master mix for all reaction tubes, plus one additional tube to allow for pipetting errors. Combine the following components in the order shown: Per rxn 40Ul dH20 5Ul 10x BioXact Long PCR buffer 1Ul 50x dNTP (10 mM) 2Ul 5’ PCR Primer IIA (10 UM) 1.5Ul MgCl2 1Ul BioXact Long polymerase 49Ul Total Volume 4. Mix by vortexing and spin tube briefly in microfuge. 5. Aliquot 49Ul of Master Mix into each reaction tube. 6. Mix contents by gently flicking tube, spin briefly in microfuge to collect contents. 7. Place tube into the preheated thermocycler, commence thermocycling using the following programme: 95°C 1 min 17 cyclesb; 95°C 5 sec 65°C 5 sec 68°C 6 min Always use heated lid and select ‘calculated’ mode when editing PCR programme. 8. Store double stranded (ds) cDNA product at -20°C prior to purification. Notes b For excellent quality RNA it is suggested that 15 to 19 cycles are tested initially. When using clinical material higher cycles may be needed. In our experience clinical RNA, even of good quality, often fails to amplify as well as cell line RNA of comparable quality. This may be related to selective mRNA degradation in the clinical samples or lower ratios of mRNA/rRNA abundance. If necessary more of the 1st strand cDNA may be added to the PCR reaction. Purification of LD PCR Using individual G-50 columns 1. Resuspend the resin in a G-50 column by vortexing gently 2. Snap off bottom closure, loosen cap a quarter turn and place column in a 1.5 ml microfuge tube 3. Pre-spin column 2000g for 1 min to remove resin buffer. Blot tip of column dry on a clean paper towel. 4. Remove top cap and place tube in a fresh 1.5ml tube. 5. Pipette the sample onto the centre of the angled surface of the resin bed being careful not to disturb the resin 6. Centrifuge 2000g for 1 min, discard the column. 7. Quantify purified (ds)cDNA LD PCR product using Spectrophotometer. 8. Store at -20°C until required. Klenow labelling using Invitrogen’s BioPrime DNA labelling system 1. Transfer a 250ng aliquot of ds cDNA to a fresh 0.2ml thin wall tube. 2. Make up to 22ml with water. 3. Add 20ml 2.5x Random Primer Reaction Buffer (Invitrogen). 4. Incubate at 95°C for 5 min, then place on ice 5. Add: 5ml 10x low-C dNTP mix (5mM dGTP, dTTP, dATP, 2mM dCTP) 2ml Cy3/Cy5 dCTPc 1ml Klenow 40U/ml (Invitrogen) 6. Incubate 37°C for 2hd (Tetrad) 7. Stop reaction by adding 5ml stop buffer (Invitrogen) 8. Wrap samples in foil to prevent Cy Dyes photo bleaching Notes c Labellings with 1 or even 0.5 Ul of Cy dye work reasonably well therefore representing considerable saving on the cost per labelling, however hybridisations may give slightly less signal. Purification of labelled targets Depending upon how many targets you have to purify choose one of the following options (normally we use individual columns if purifying less than 10 targets); Using individual G-50 columns 9. Resuspend the resin in a G-50 column by vortexing gently 10. Snap off bottom closure, loosen cap a quarter turn and place column in a 1.5 ml microfuge tube 11. Pre-spin column 2000g for 1 min to remove resin buffer. Blot tip of column dry on a clean paper towel 12. Remove top cap and place tube in a fresh 1.5ml tube. 13. Pipette the sample onto the centre of the angled surface of the resin bed being careful not to disturb the resin (purify Cy3 and Cy5 separately) 14. Centrifuge 2000g for 1 min, discard the column. 15. Pool the purified Cy3 and Cy5 labelled products in a clean 1.5ml tube. 16. Add 1/10th volume of 3M NaOAc, pH 5.2 and 2.5 volumes of 100% EtOH 17. Mix by vortexing; incubate at -70oC for 30 min, then centrifuge 13K rpm at 4oC for 20 minutes. 18. Remove EtOH being careful not to disturb the pellet, which should be clearly visible 19. Wash pellet with 750ml of 70% EtOH, centrifuge 13K rpm for 5 min. 20. Remove washings using 10ml pipette tip and air dry in the dark for 20-30 min. 21. Store at -20°C until required. Target preparation 1. For each sample re-suspend the pellet from the co-precipitated samples in 40ml of Hybridization buffer (see recipe below) vortexing if necessary. 2. Remove 1.5ml into a clean tube for quantification (see below). 3. Heat targets at 95°C for 5 min. 4. Place on microarray. Hybridization buffer 50ml - 40% deionised formamide 20ml of 100% - 5 x Denhart’s 5ml of 50x stock - 5 x SSC 12.5ml of 20x stock - 1mM Na pyrophosphate 0.5ml of 100mM stock - 50mM Tris pH 7.4 2.5ml of 1M stock - 0.1% SDS 0.5ml of 10% stock 9.0ml H2O Filter through 0.22um filter and allow to warm to RT before use to allow SDS to go back into solution. (Parameters: Amount of nucleic acid labeled = 500, Amplification = PCR, Mass unit = Nano gram)
Animal collection and maintenance Specimens of L.elliptica were collected by scuba divers at a depth of 10-18 m in February 2006 at North Cove, Rothera Point, Adelaide Island, Antarctic Peninsula (67°34 07 S, 68°07 30 W). Clams were shipped to the British Antarctic Survey facilities in Cambridge, UK and allowed to acclimate to laboratory conditions for four weeks before the experiments were carried out. During the acclimation period, animals were kept in plastic meshed baskets suspended in a 500 l tank with a water circulating system. Water temperature and salinity were stable at 0±0.5°C and 32-36 p.p.t. respectively. Individuals were exposed to a L:D cycle of 12 h:12 h throughout the four weeks. A marine microalgae concentrate (Reed Mariculture, Campbell, California, USA) was added to the water on a weekly basis. No mortalities were recorded during the acclimation period. Heat stress exposure At the end of the acclimation period, eight individuals were transferred to a 60 l jacketed water bath with aerated seawater connected to a thermo-circulator (Grant LTD 20G, Grant Instruments LTD, Cambridge, UK). Seawater temperature was raised from 0 C to 3 C ± 0.5 C over a 12 h period. The temperature was maintained constant for another 12 h. Following exposure, specimens were individually removed from the experimental tank and tissue samples were taken from the mantle, siphon and gill. Samples were snap frozen in liquid nitrogen and stored at -80ºC. The procedure was repeated for a control group with eight specimens, for which seawater temperature was maintained constant at 0 C ± 0.5 C. Experiments were carried out at the same time of the day in order to exclude potential differences as a result of daily patterns in metabolism. There were no mortalities or whole animal indicators of stress in either group.
Hybridizations carried out under cover slips in a humid chamber. (Parameters: Chamber type = OTHER: Grant Boekel Hybridization chamder, Quantity of label target used = 5, Mass unit = Micro gram, time = 18, Tiny time unit = hours, Volume = 20, Volume unit = Micro litre, temperature = 49)
RNA preparation. Total RNA was extracted on ice by homogenising ~50 mg of tissue samples in 600 µl Tri Reagent (Sigma) using glass homogenisers. Immediately after, 120 µl chloroform was added to the homogenate, which was mixed vigorously and centrifuged at 13000g for 15 min at 4 C. After centrifugation, the upper aqueous phase was transferred into a fresh 1.5 ml tube to which 150 µl isopropanol and 150 µl saline solution (1:1 0.8 M sodium citrate and 1.2 M NaCl) was added. After a second 10 min centrifugation at the same speed and temperature, the supernatant was discarded and the RNA pellet washed with 100 µl 75% ethanol. Following a 1 min centrifugation, the ethanol was removed and, after drying, the pellet was resuspended in 250 µl DEPC treated water. An equal volume of 7.5 M LiCl was added to the sample, incubated at -80 C for 30 min and centrifuged at 13000g for 20 min at 4 C. The supernatant was removed and the pellet was washed with ethanol, air dried and dissolved in 50 µl DEPC treated water. RNA concentrations were estimated using a Nanodrop spectrophotometer (ND-1000, Thermo Scientific). Samples for RNA amplification and labelling were cleaned using RNeasy mini kit columns (Qiagen) following manufacturer instructions in order to eliminate LiCl and salt residues. (Parameters: Extracted product = total_RNA, Amplification = PCR)
RNA was extracted from each individual following the modified Tri Reagent method specified. Equal amounts of RNA from each individual were then pooled together.