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Sample GSM588425 Query DataSets for GSM588425
Status Public on Sep 01, 2010
Title 73-70
Sample type RNA
 
Channel 1
Source name Challenge Infection week 2 Pool
Organism Ovis aries
Characteristics tissue: Afferent Lymph collected over a 12 week period from sheep 1001-6
strain: up to 5 pen raised, nematode naïve, outbreed Romney sheep
Biomaterial provider AgResearch NZ
Treatment protocol Challenge Infection week 2 Pool
Growth protocol Lymph cells were collected daily, RNA extracted and pooled for each weekly period for all sheep
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using Trizol® Reagent (Invitrogen Auckland, NZ), a modification of the acid guanidinium thiocyanate-phenol-chloroform extraction method according to the manufacturers’ protocol and incorporated the optional step (for tissues with high content of protein, fat, polysaccharides) during sample homogenization. The appropriate RNA samples were pooled and further purified using an RNeasy kit (Qiagen, Hilden, Germany).
Label Cy5
Label protocol Fluorescent-labeling reactions used the SuperScript Indirect cDNA Labeling System (Invitrogen, Auckland, NZ). cDNA synthesised from 20µg total appropriately pooled RNA was labeled in the presence of either mono-functional N-hydroxysuccinimide (NHS)-ester Cy5 or Cy3 dyes (Amersham Biosciences, Piscataway, NJ, USA). Labeled cDNAs were purified to remove unincorporated dyes using the supplied SNAP columns, combined and concentrated to 10 µL by ethanol precipitation.
 
Channel 2
Source name Challenge Infection week 3 Pool
Organism Ovis aries
Characteristics tissue: Afferent Lymph collected over a 12 week period from sheep 1001-6
strain: up to 5 pen raised, nematode naïve, outbreed Romney sheep
Biomaterial provider AgResearch NZ
Treatment protocol Challenge Infection week 3 Pool
Growth protocol Lymph cells were collected daily, RNA extracted and pooled for each weekly period for all sheep
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using Trizol® Reagent (Invitrogen Auckland, NZ), a modification of the acid guanidinium thiocyanate-phenol-chloroform extraction method according to the manufacturers’ protocol and incorporated the optional step (for tissues with high content of protein, fat, polysaccharides) during sample homogenization. The appropriate RNA samples were pooled and further purified using an RNeasy kit (Qiagen, Hilden, Germany).
Label Cy3
Label protocol Fluorescent-labeling reactions used the SuperScript Indirect cDNA Labeling System (Invitrogen, Auckland, NZ). cDNA synthesised from 20µg total appropriately pooled RNA was labeled in the presence of either mono-functional N-hydroxysuccinimide (NHS)-ester Cy5 or Cy3 dyes (Amersham Biosciences, Piscataway, NJ, USA). Labeled cDNAs were purified to remove unincorporated dyes using the supplied SNAP columns, combined and concentrated to 10 µL by ethanol precipitation.
 
 
Hybridization protocol Micro array slides were prehybridized in a preheated (42oC) 0.22 µm filtered solution of 5 x SSC, 0.1% SDS and 0.25% BSA (A-788; Sigma Chemical company, St. Louis, MO, USA) for 20 min at 42oC. Slides were rinsed in deionised water then isopropanol and air dried before hybridization. cDNA labeled samples were heat-denatured (95oC for 5 min) and mixed with 60 µL of pre-warmed (68oC) SlideHyb No1 (Ambion, Austin, TX, USA) added. The final cDNA labeled solution was loaded onto the slides with lifter coverslips (Erie Scientific, Portsmouth, NH, USA). Hybridizations were conducted for 20 h at 52oC in sealed humidified chambers (CMT Hybridization Chambers, Corning, NY, USA). Following hybridization, slides were washed for 5 min each in (1) 2 x SSC, 0.1% SDS, (2) 1 x SSC and (3) 0.1 x SSC then centrifuged at 1000 g for 5 min
Scan protocol The slides were stored in the dark to minimize photo bleaching, and scanned for both dye channels at 532 and 635 nm within two days using an GenePix Professional 4200A scanner (Molecular Devices, Sunnyvale, CA, USA) with a 10-μm resolution. The photomultiplier tube voltage was adjusted to obtain an equal global intensity in both channels.
Description Afferent Lymph was collected from up to 5 pen raised, nematode naïve, outbreed Romney sheep (Tag No’s 1001, 1002, 1003, 1004, 1006). During this time a Pre-Infection sample was collected, the sheep were immunised by 3 truncated (2 week) T. colubiformis infections (40,000 Tc) followed by a Challenge Infection with 40,000 T. colubiformis L3 (Challenge Infection weeks 1-3). The Challenge Infection will enable us to determine changes in gene expression when the sheep gut rejects the parasitic nematodes.
Data processing The normalization method used as in PubMed ID 15231532 [Bioinformatics. 2004 Nov 22;20(17):3196-205].
 
Submission date Aug 30, 2010
Last update date Aug 31, 2010
Contact name Alan Francis McCulloch
E-mail(s) alan.mcculloch@agresearch.co.nz
Phone 64 3 4899080
Organization name AgResearch NZ
Street address Invermay
City Puddle Alley
State/province Mosgiel
ZIP/Postal code n/a
Country New Zealand
 
Platform ID GPL4077
Series (2)
GSE23863 Regional Immune Response to Trichostrongylis colubriformis - Gene Expression during Challenge of Immunized Sheep
GSE23890 Regional Immune Response to Trichostrongylis colubriformis in Sheep

Data table header descriptions
ID_REF
F1MEAN Channel 1 raw foreground mean
F2MEAN Channel 2 raw foreground mean
B1MEAN Channel 1 raw background mean
B2MEAN Channel 2 raw background mean
VALUE Log10(Cy5/Cy3)
NORMCH1 Normalised Cy5 value
NORMCH2 Normalised Cy3 value

Data table
ID_REF F1MEAN F2MEAN B1MEAN B2MEAN VALUE NORMCH1 NORMCH2
285308 554 622 322 510
285282 349 463 313 503
285256 302 473 309 514
285230 690 638 294 498
285204 395 521 299 475
285178 14998 11292 308 490 .05 13454 11956
285152 1647 730 324 526
285126 328 466 339 507
285100 283 499 325 526
285074 355 569 309 532
285048 6927 5483 346 524 .04 6067 5534
285022 21505 18888 333 555 -.06 18557 21084
284996 10011 7433 331 527 .04 8645 7904
284970 18651 12787 342 631 .07 16333 13917
284944 23799 18122 348 685 .01 20698 20115
284918 48005 33770 347 586 .04 41738 38132
284892 60478 52189 340 643 -.04 53185 58594
284866 7673 6156 319 649 .04 6847 6196
284840 39172 33055 309 500 -.05 33729 37669
284814 13085 8742 323 547 .11 11673 9106

Total number of rows: 19968

Table truncated, full table size 690 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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