13 protocols
AccessionType
feature_extraction
Scanning Slide scanning 1. Turn power on scanner and restart the PC (so the PC can recognise the scanner). 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 micrometres, and Lines to average to 2. 5. Start a low resolution scan by clicking the Preview Scan button; 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 two single image .tiff files (one for each channel) by using the Open/Save function. We save the images after the batch and slide number printed on the microarray label.
hybridization
20 ug of lcts were hybridised to microarray slides in 48% formamide at 55 C for 16-20 hrs, in a humid chamber. The slides were then washed at RT.
labeling
Labelling of 20ug mRNA was performed without prior amplification to avoid introducing bias due to transcript abundance, length and GC content. The cDNAs were generated by reverse transcription of total RNA using Superscript III enzyme (Invitrogen), primed with anchored oligo-dT23 (Sigma Aldrich, Gillingham, UK), and labelled with dNTPs incorporating either a dCTP Cy3 or Cy5 dye (Perkin Elmer, Beaconsfield, UK). Labelled products were purified using AutoSeq G50 columns (Amersham Biosciences)
labeling
Labelling of 20ug mRNA was performed without prior amplification to avoid introducing bias due to transcript abundance, length and GC content. The cDNAs were generated by reverse transcription of total RNA using Superscript III enzyme (Invitrogen), primed with anchored oligo-dT23 (Sigma Aldrich, Gillingham, UK), and labelled with dNTPs incorporating either a dCTP Cy3 or Cy5 dye (Perkin Elmer, Beaconsfield, UK). Labelled products were purified using AutoSeq G50 columns (Amersham Biosciences)
nucleic_acid_extraction
If necessary samples were split into aliquots containing no more than 100mg of parasite material, 1ml of Trizol (Invitrogen) was added to each sample and homogenized with a glass Teflon homogenizer. Samples were incubated for five minutes at room temperature. After incubation 200ul of chloroform was added to each sample, shaken vigorously for 15 seconds, incubated at room temperature for a further three minutes and then centrifuged for 15 minutes in a chilled centrifuge (4 C ) at 12,000ug. After centrifugation the top aqueous phase was transferred to a new microfuge tube, 0.5ml of isopropanol and 1ul of Glycoblue (Ambion) were added, mixed and incubated at room temperature for ten minutes. The RNA pellet was sedimented by centrifugation for 30 minutes in a chilled centrifuge at 12,000ug and then washed by removing the liquid phase by aspiration and replacing with 1ml of 75% ethanol. The 75% ethanol wash was vortexed, spun again in a chilled centrifuge at 12,000ug for 15 minutes, then removed by aspiration. The RNA pellet was air dried before re-suspension in 300ul of nuclease free water, and quantified but 260/280 spectral analysis.

RNA was precipitated under ethanol, for storage, by addition of 30ul of 3M sodium-acetate pH5.2 and 990ul of 100% ethanol and left overnight at -80 C. The RNA pellet was sedimented by centrifugation for 30 minutes in a chilled centrifuge at 12,000ug and then the liquid phase was replaced with 1ml of 75% ethanol before returning to -80 C for long term storage.
specified_biomaterial_action
Adult and 21 day worms were obtained by portal perfusion of infected NMRI strain mice eight weeks and 21 days after infection, respectively. Mice used as hosts for adult worms were infected with 200 cercaria via the abdominal skin, while mice for 21 day worms were infected with 250 cercaria via the abdominal skin.
grow
All samples are derived from the Puerto Rican isolate of S. mansoni and the life cycle maintained in the snail Biomphalaria glabrata and the NMRI strain of mice.
pool
A control pool was created by combining equal amounts of total RNA from the 7 life cycle stages.
specified_biomaterial_action
Daughter sporocysts were obtained by dissection in 0.5x PBS, with the aid of a dissecting microscope, from the hepatopancreas of snails infected 21 days previously with 40 miracidia each. The germ balls were separated from host snail tissue using a drawn out glass pipette and collected in a chilled watch glass.
specified_biomaterial_action
Cercariae were obtained from snails previously infected by exposure to 10 miracidia each. Shedding was induced by placing infected snails in the dark for 48 hours, after which they were transferred to 100ml glass beakers containing approximately 25ml of pond water before being exposed to bright light for two hours.
specified_biomaterial_action
NMRI strain mice were culled eight weeks post infection with 200 cercaria. Livers were removed and macerated in a Waring blender in mM Na2HPO4 3.3mM KH2PO4. The liver homogenate was incubated at 37 C in a shaking water bath with trypsin before filtration through a 180um mesh sieve; eggs sink to the bottom of the collecting dish where they can be collected and washed with 0.9% saline.
specified_biomaterial_action
The pond water containing cercariae was collected in a 500ml glass beaker and chilled on ice for one hour to concentrate cercariae by sedimentation. Excess pond water was removed by aspiration. All of the following was performed under sterile conditions in a filter cabinet, observing the appropriate aseptic techniques. Cercariae were sedimented by pulse centrifuging down from suspension in round-bottomed 10ml centrifuge tubes at 250ug. Supernatant was removed and the cercarial pellet washed with 10ml RPMI M169 w/o glutamine (Gibco BRL). The cercariae were re-pelleted by a second centrifugation (250ug) and resuspended in 1ml of RPMI with 1% penicillin/streptomycin (pen/strep, 10,000IU/ml and 10mg/ml stock respectively).

The cercariae were vortexed for 2 minutes to remove their tails and transform them into the skin-penetrating schistosomula. The mixture was then transferred to a 40ml cell culture flask, topped up to 25-30ml with 1% pen/strep RPMI and left to incubate at 37 C 5% CO2 for 3 hours. After incubation the supernatant was removed leaving the schistosomula and tails in ~5ml of solution at the bottom of the flask. The sediment was then resuspended and carefully layered on a prepared discontinuous RMPI/percoll gradient (70%/40%). Separation was then performed by centrifugation for 12 minutes at 300ug. The schistosomula were recovered by carefully pipetting off the 70%/40% interface into clean 10ml round-bottomed centrifuge tubes. These were then washed 8 times in 10ml of RPMI, recovering the schistosomula by pulse-centrifugation of up to 250ug with each wash. The final pellets were resuspended in ~10ml of 1% penicillin, streptomycin and glutamine (from 200mM stock) RPMI, supplemented with 10% FCS and using a pastette drop-pipetted into 24 well plates containing 1ml of the same medium. The plates were incubated at 37C 5% CO2 for the appropriate time before recovery by transfer to 50ml centrifuge tubes and centrifugation at 60ug.
unknown_protocol_type
Reagents

Amine binding slides (3-D link activated slides, Motorola, DN01-0025)
DNA elements in 96-well thin-walled thermowell plates
humid chamber(NaCl saturated water in an air-tight container)
1% w/v ammonium hydroxide solution (made in Milli-Q dd H20)
0.1% w/v sodium dodecyl sulphate solution (made in Milli-Q dd H20)
Blocking solution: 50mM ethanolamine, 0.1M Tris(Ph9)
Post-coupling wash solution: 4xSSC, 0.1% SDS
Milli-Q 18.2MWcm ddH20 (Milli-Q plus 185 purification system)
microscope slide rack
microscope slide storage boxes
centrifuge (Sorvall RT 6000D orequivalent)

Method

1. Array DNA elements onto codelink amino binding slidesa at 20-25C, 40-50% relative humidity.

2. Transfer slides containing arrayed elements into a microscope slide rack and place in a humid chamber and incubate for 24-72 hours at room temperature.

3. Remove from humid chamber and place in rack. Shake in pre-warmed blocking solution at 50C for 30 minutes.

4. discard blocking solution

5. rinse slides twice with Milli-Q water

6. Wash the slides with 10 ml post-coupling solution (pre-warmed to 50C) for 30 minutes on the shaker.

7. Discard the wash solution and wash twice with Milli-Q water

8. place the slides in bioling water for 2 minutes and agitate

9. Place slides in the rack and centrifuge at 1200rpm for 4 minutes

10. Place slides in a storage box and store at room temperature in a cool, dry place until used.