Comment[ArrayExpressAccession] E-MEXP-3453 Investigation Title Seasonal and spatial influences on gene expression in Antarctic krill. Comment[Submitted Name] Seasonal and spatial influences on gene expression in Antarctic krill. Comment[AEExperimentDisplayName] Seasonal and spatial influences on gene expression in Antarctic krill. Comment[MIAMExpressLogin] paulseear7 Comment[MIAMExpressSubmissionID] 7942 Experiment Description Effect of season and winter lattitude on gene expression in Antarctic krill. Experimental Design reference_design co-expression_design transcript_identification_design Comment[AEExperimentType] transcription profiling by array Experimental Factor Name GeographicLocation Experimental Factor Type sampling_site Person Last Name Seear Person First Name Paul Person Email ps255@le.ac.uk Person Phone 0044 116 252 3350 Person Affiliation University of Leicester Person Address Biology, Adrian Building, University Road,, Leicester, Leicester, LE1 7RH, United Kingdom Person Roles submitter Person Roles Term Source REF MGED Ontology Quality Control Type biological_replicate Public Release Date 2012-11-01 Comment[ArrayExpressSubmissionDate] 2011-11-25 17:16:03 Publication Status not yet submitted Protocol Name P-MTAB-24136 P-MTAB-24137 P-MTAB-24138 P-MTAB-24139 P-MTAB-24140 P-MTAB-24141 P-MTAB-24142 Protocol Description Hybridised slides were scanned at 10 ?m resolution

using a GenePix 4100 microarray scanner (MDS technologies).

Image analysis and local background correction

of spot intensities were then performed with

GenePix Pro 6.0 software (MDS technologies). Array

images were only used if the following quality thresholds

were passed: median signal-to-background >8; median

signal-to-noise >10 and mean of median background

signal <250. Anomalous spots were flagged and

excluded from future analyses. GenePix results files

were then imported into GeneSpring GX 11.1 (Agilent

Technologies) for further analysis.
(Parameters: Scanning hardware = GenePix Personal 4100A [Axon Instruments], Scanning software = GenePix Pro [Axon Instruments]) Postlarval krill were sampled at two main locations, one close to the Antarctic Peninsula and the other at South Georgia by the RRS James Clark Ross (JCR) and the krill fishery vessel Saga Sea. The krill were caught with target net tows in the top 200 m. On the JCR cruise, they were placed immediately in RNA later. On the fishery vessels, they were immediately deep frozen to -20oC and stored over the longer term at -80oC.

In the laboratory, the heads were taken for RNA extraction while the remainder of the body tissue was used for measurement uropod length, sex, sexual stage and moult stage. Studies of transcriptional expression were limited to those individuals in the intermoult stage (stage C) that were female but yet to fully mature.
(Parameters: time unit = seconds, temperature unit = C) Required Reagents

Use HPLC-purified primers, their sequences are as follows;

3M-^R SMART CDS primer IIA

(5M-^R-AAGCAGTGGTATCAACGCAGAGTAC-T30VN-3M-^R)

SMART IIA Oligonucleotide

(5M-^R-AAGCAGTGGTATCAACGCAGAGTACGC)r(GGG)-3M-^R)

5M-^R PCR Primer IIA (5M-^R-AAGCAGTGGTATCAACGCAGAGT-3M-^R)

Use PrimeScript RT (Takara #826801) and Advantage 2 polymerase mix (Takara #639201)

M-^U 0.2 ml thin wall tubes

illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare #28-9034-71)

M-^U BioPrime DNA labelling system (Invitrogen #18094-011)

M-^U 10x low-C dNTP mix (5mM dGTP, dTTP, dATP, 2mM dCTP) (Sigma #DNTP-100A)

M-^U illustra MicroSpin G-50 columns (GE Healthcare #27-5340-02)

M-^U Cy3-dCTP and Cy5-dCTP (GE Healthcare #PA53023 and PA55023, respectively)

M-^U Ethanol

M-^U 3M Sodium Acetate

M-^U Nuclease-free water

1st Strand cDNA Synthesis

1. Preheat Tetrad to 72M-0C.

2. For each sample, combine the following reagents in a sterile, thin wall 0.2 ml tube:

1M-5l total RNA (500ng/M-5l stock)

1M-5l 3M-^R SMART CDS Primer IIA (10 M-5M)

1M-5l SMART IIA Oligonucleotide (10 M-5M) stored at -70M-:

1M-5l Stratagene Alien Spike RNA

1M-5l dH2O

5M-5l Total volume

3. Mix contents and spin the tube briefly in a micro centrifuge.

4. Incubate at 72M-0C 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:

2M-5l 5x First-strand buffer

1M-5l DTT (0.1 M)

1M-5l dNTP (10 mM)

1M-5l PrimeScript RT

8. Mix by gentle pipetting and spin tubes briefly in microphage.

9. Incubate the tubes at 42M-0C for 1 hr (in Tetrad, use heated lid).

10. Place tubes on ice to terminate first-strand synthesis.

cDNA Amplification by LD PCR

1. Transfer a 1M-5l 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 M-^V20oC.

2. Preheat Tetrad to 95M-0C.

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

40M-5l dH20

5M-5l 10x Advantage 2 PCR buffer

1M-5l 50x dNTP (10 mM)

2M-5l 5M-^R PCR Primer IIA (10 M-5M)

1M-5l Advantage 2 polymerase mix

49M-5l Total Volume

4. Mix by vortexing and spin tube briefly in microfuge.

5. Aliquot 49M-5l 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:

95M-0C 1 min

19 cycles;

95M-0C 5 sec

65M-0C 5 sec

68M-0C 6 min

Always use heated lid and select M-^QcalculatedM-^R mode when editing PCR programme.

8. Store double stranded (ds) cDNA product at -20M-0C prior to purification.

9. Purify LD PCR products using individual illustra GFX columns following manufacturerM-^Rs instructions.

Klenow labelling using InvitrogenM-^Rs 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 distilled water (Invitrogen).

3. Add 20ml 2.5x Random Primer Reaction Buffer (Invitrogen).

4. Incubate at 95M-0C 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 37M-0C for 2hd (Tetrad)

7. Stop reaction by adding 5ml stop buffer (Invitrogen)

8. Wrap samples in foil to prevent Cy Dyes photo bleaching

Purification of labelled 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 -20M-0C until required.

Target preparation

1. For each sample re-suspend the pellet from the co-precipitated samples in 42ul of Hybridization buffer (see recipe below) vortexing if necessary.

2. Add 3 ug Poly A+ DNA and 3 ug yeast tRNA

3. Heat targets at 95M-0C for 5 min, vortexing every minute and cool at room temperature.

4. Place on microarray.

Hybridization buffer

50ml

- 40% deionised formamide 20ml of 100%

- 5 x DenhartM-^Rs 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.

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).
(Parameters: Amount of nucleic acid labeled = 500, Amplification = PCR, Mass unit = Nano gram) GenePix results files were imported into GeneSpring GX 11.1 (Agilent Technologies). Normalisation was performed using a M-^QPer Spot and Per ChipM-^R intensity dependent (Lowess) normalisation using software defaults (20% smoothing/cutoff 10). Hybridizations carried out under cover slips in a humid chamber.
(Parameters: Chamber type = OTHER: Grant Boekel Hybridization chamder, Quantity of label target used = 1, Mass unit = Micro gram, time = 18, Tiny time unit = hours, Volume = 40, Volume unit = Micro litre, temperature = 49) Pooled 0.5ug amplified cDNA (see BAS SMART labelling) from all individuals to serve as pooled reference sample. Total RNA was extracted from the frozen heads of krill (including eyes and brain) using TriSure (Bioline) followed by purification with RNeasy columns (Qiagen). Concentration and purity of the total RNA was determined using a Nanodrop spectrophotometer (LabTech International), while degradation was checked by electrophorescing one microgram of total RNA on a non-denaturing 1.5% (w/v) agarose gel.
(Parameters: Extracted product = total_RNA, Amplification = none) Protocol Type image_acquisition grow labeling bioassay_data_transformation hybridization pool nucleic_acid_extraction Protocol Term Source REF MGED Ontology MGED Ontology MGED Ontology MGED Ontology MGED Ontology MGED Ontology MGED Ontology Term Source Name MGED Ontology Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php SDRF File E-MEXP-3453.sdrf.txt PubMed ID 22802932 Publication Author List Seear PJ, Goodall-Copestake WP, Fleming AH, Rosato E, Tarling GA Publication Title Seasonal and spatial influences on gene expression in Antarctic krill Euphausia superba Publication DOI doi:10.3354/meps09947