Affymetrix GeneChip Rat Genome U34C [RG_U34C]

Affymetrix GeneChip Rat Genome U34B [RG_U34B]

Affymetrix GeneChip Rat Genome U34A [RG_U34A]

Dissection of Chromosome 18 Blood Pressure and Salt-Sensitivity Quantitative Trait Loci in the Spontaneously Hypertensive Rat

rawData

scan

processedDataMatrix

Four male SHR/Ola, BN and SHR-18 rats were fed a normal diet and sacrificed at 9 weeks of age. Four male SHR/Ola and SHR-18 rats at 8 weeks of age were fed 1% NaCl for one week and then sacrificed. Kidneys were removed and frozen in liquid nitrogen for all 20 animals. Total RNA was isolated, labelled cRNA was generated and hybridised to Affymetrix Rat RG-U34ABC arrays.

Nutrients

StrainOrLine

Transcription profiling of kidney from rats of SHR/Ola, BN and SHR-18 strains after being provided with drinking water with 1% or 0% sodium chloride

Four male SHR/Ola, BN and SHR-18 rats were fed a normal diet and sacrificed at 9 weeks of age. Four male SHR/Ola and SHR-18 rats at 8 weeks of age were fed 1% NaCl for one week and then sacrificed. Kidneys were removed and frozen in liquid nitrogen for all 20 animals. Total RNA was isolated, labelled cRNA was generated and hybridised to Affymetrix Rat RG-U34ABC arrays.


csc.mrc.ac.uk:mimir/Protocol:CWTA_RNA_concentrating

RNA pools were concentrated to 2ug/ul by ethanol precicipation. The pools were added to an 1.5ml eppendorf tube containing 2 x volume of 100% ethanol and 1/10 volume of sodium acetate. Tubes were vortexed and allowed to stand at room temperature for 15 minutes. The samples were then centrifuged in a bench-top microfuge at maximum speed for 20 minutes. The supernantant was removed, and the pellet washed with 70% ethanol. Following vortexing, centrifugation and removal of the supernantant, the pellet was air-dried at room temperature. Pellets were resuspended in 80ul DEPC-treated water.

csc.mrc.ac.uk:mimir/Protocol:CWTA_RNA_isolation

csc.mrc.ac.uk:mimir/Protocol:CWTA_kidney_removal

Kidneys were removed and snap frozen. Pairs of whole kidneys were ground to a fine powder in liquid nitrogen with pestle and motar.

csc.mrc.ac.uk:mimir/Protocol:cRNA_frag

csc.mrc.ac.uk:mimir/Protocol:cRNA_prep_affy

csc.mrc.ac.uk:mimir/Protocol:hyb

Hybridisation of fragmented cRNA  Things to note: 1. There are two septa on the backside of the GeneChip.  Whilst filling or emptying the hybridisation chamber through one septum, a pipette tip must be inserted into the other as a vent. 2. The more you inset/remove tips into the septa, the looser they will become.  Eventually they will start to leak.  Method 1. Equilibrate array at room temperature for 30 minutes before use. 2. Heat frozen fragmented cRNA at 99oC for 5 minutes. 3. During incubation, pre-wet the array by filling with 200ul (100ul for test or mini arrays) of 1x hybridisation Buffer.  Incubate in rotisserie oven at 45oC, 60rpm for 10 minutes. 4. Cool fragmented cRNA at 45oC for 5 minutes. 5. To bring any particulate matter to the bottom of the tube, spin fragmented cRNA at maximum speed (?13,000g) for 5 minutes. 6. Remove buffer solution from array, ensuring that all solution is removed. 7. Add 200ul (100ul for test or mini array) of fragmented cRNA avoiding any debris at the bottom of the tube. 8. Hybridise in rotisserie oven at 45oC, 60rpm, for 16 hours (overnight). 9. Store remaining volume of fragmented cRNA at –20oC. 10. After hybridisation, proceed to “Preparation of GeneChip station”.  

Transcription profiling of kidney from rats of SHR/Ola, BN and SHR-18 strains after being provided with drinking water with 1% or 0% sodium chloride

Submitter Supplied Information

Samples