8 protocols
AccessionType
feature_extraction
Title: Affymetrix CEL analysis. Description:
bioassay_data_transformation
Title: Affymetrix CHP Analysis (ExpressionStat). Description:
nucleic acid labeling protocol
Labeling of samples was carried out according to the manufacturer’s standard protocol
hybridization
Title: Affymetrix EukGE-WS2 Hybridization. Description:
nucleic_acid_extraction
Trizol (Invitrogen, 1ml) was applied directly to the bead-bound vessels and total RNA was extracted according to the manufacturer’s instructions. RNA was purified with an RNeasy Mini Kit (Qiagen) and concentrated using a Speed Vac to 33 ng/ul. Affymetrix Two-Cycle Target labelling kit was then used, according to manufacturer’s instructions, starting with 100 ng total RNA. (Parameters: Extracted product = total_RNA, Amplification = RNA polymerases)
pool
C57BL/6J mouse: 12 retinae from 6 mice pooled prior to vessel isolation and RNA extraction (per chip). RD1 mouse: 12 retinae from 6 mice pooled prior to vessel isolation and RNA extraction (per chip). Grh13ct (curlytail) mouse : 12 retinae from 6 mice pooled prior to vessel isolation and RNA extraction (per chip). VLDLR knock-out mouse: 12 retinae from 6 mice pooled prior to vessel isolation and RNA extraction (per chip). RCS non-dystrophic rat: 6 retinae from 3 rats pooled prior to vessel isolation and RNA extraction (per chip). RCS dystrophic rat: 6 retinae from 3 rats pooled prior to vessel isolation and RNA extraction (per chip).
specified_biomaterial_action
Animals were used at the following ages: C57BL/6J – 15 weeks,RD1 mouse – 18 weeks, Grh13ct (curlytail) – 13 weeks, VLDLR-/- mouse – 16 weeks, RCS non-dystrophic rat – 20 weeks, RCS dystrophic rats - 20 weeks. Mice were euthanased by cervical dislocation and rats were killed by CO2 exposure, followed by cervical dislocation. Retinae where immediately removed. Six rat retinae or 12 mouse retinae were enzymatically digested at 37 oC for 45-60 min with periodic trituration. Retinal vessel fragments were obtained by incubation with a PECAM-1 antibody that was conjugated to magnetic beads. Bead-bound vessels were separated from the vessel-depleted fraction using the magnetic seperator. The vessel-enriched fractions were used for RNA extraction.
grow
Husbandry conditions: Temperature: 18-21 degrees C, Humidity: 55% +/-10%, light-dark cycles: 12h, water: tap water, nutrients: Housing: conventional caging compliant with Home Office Guidelines. Parameters: time unit = seconds, temperature unit = C)