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 1 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.
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].
