Affymetrix GeneChip Mouse Expression Array MOE430A [MOE430A]

Affymetrix GeneChip Mouse Expression Array MOE430B [MOE430B]

Affymetrix GeneChip Murine Genome U74Av2 [MG_U74Av2]

Kidney-specific upregulation of vitamin D3 target genes in ClC-5 KO mice

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Mutations in ClC-5 cause Dent's disease, a disorder associated with low molecular weight proteinuria, hyperphosphaturia and kidney stones. ClC-5 is a Cl /H+-exchanger predominantly expressed in the kidney, where it facilitates the acidification of proximal tubular endosomes. The reduction in proximal tubular endocytosis resulting from a lack of ClC-5 raises the luminal concentration of filtered proteins and peptides like PTH. We used gene expression profiling to identify possible signaling pathways that might be changed in ClC-5 KO kidneys, bones and intestines. Mouse model described in Piwon et al, ClC-5 Cl--channel disruption impairs endocytosis in a mouse model for Dent's disease, Nature 408, 369-373 (16 November 2000),doi: 10.1038/35042597

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organism part

Transcription profiling by array of mouse Clcn5 knockout in kidney, bone and intestine

Mutations in ClC-5 cause Dent's disease, a disorder associated with low molecular weight proteinuria, hyperphosphaturia and kidney stones. ClC-5 is a Cl /H+-exchanger predominantly expressed in the kidney, where it facilitates the acidification of proximal tubular endosomes. The reduction in proximal tubular endocytosis resulting from a lack of ClC-5 raises the luminal concentration of filtered proteins and peptides like PTH. We used gene expression profiling to identify possible signaling pathways that might be changed in ClC-5 KO kidneys, bones and intestines.<br%gt; Mouse model described in Piwon et al, ClC-5 Cl--channel disruption impairs endocytosis in a mouse model for Dent's disease, Nature 408, 369-373 (16 November 2000),doi: 10.1038/35042597

Transcription profiling of mouse KO Clcn5-/- model for Dents disease in kidney, bone and intestine

Mutations in ClC-5 cause Dent's disease, a disorder associated with low molecular weight proteinuria, hyperphosphaturia and kidney stones. ClC-5 is a Cl /H+-exchanger predominantly expressed in the kidney, where it facilitates the acidification of proximal tubular endosomes. The reduction in proximal tubular endocytosis resulting from a lack of ClC-5 raises the luminal concentration of filtered proteins and peptides like PTH. We used gene expression profiling to identify possible signaling pathways that might be changed in ClC-5 KO kidneys, bones and intestines. Mouse model described in Piwon et al, ClC-5 Cl--channel disruption impairs endocytosis in a mouse model for Dent's disease, Nature 408, 369-373 (16 November 2000),doi: 10.1038/35042597

Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4

Title: Fluidics Station Protocol. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

Total RNA was prepared from the respective organ by immediate transfer of the frozen pulverized tissue into TRIZOL (1ml Trizol per 70 mg of tissue)(Gibco BRL) and homogenized using a mixer. The homogenized tissue was incubated in TRIZOL at room temperature for 5 min before addition of chloroform (0.2 x vol TRIZOL). The mixture was shaken vigorously for 15 sec and incubated at room temperature for 3 min before centrifugation for 10 min at 4C with 10000 rpm. The upper phase  was transferred carefully into a fresh microtube and isopropanol was added. After vortexing, the tube was incubated for 10 min at room temperature before centrifugation at 14000 rpm, 4C, for 15 min. The RNA pellets were washed twice with 75% EtOH and dried at room temperature before resuspending in 100 µl DEPC-treated water. The concentration and purity of the RNA was determined by optical density measurement at 260 nm and 280 nm. An aliquot of the RNA (1-2 µg) was analysed by TAE-agarose gel electrophoresis. The total RNA was then further purified using RNeasy columns according to the manufacturer's protocol (Qiagen) and eluted with 35-40 µl DEPC-treated water. Preparation of cDNA from total RNA, 15 µg of purified total RNA were used to prepare cDNA using the Invitrogen double-stranded cDNA kit (# 11917-010). Briefly, the RNA was annealed with 100 pmol HPLC-purified T7(dT)24 primer (GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-TTTTTTTTTTTTTTTTTTTTTTTT) at 70C for 10 min, quick-chilled on ice and centrifuged briefly. On ice, 4 µl 5x reaction buffer, 2 µl 0.1 M DTT and 1 µl 10 mM dNTPs were added and incubated at 42C for 2 min to equilibrate the temperature. 2 µl SuperScriptTM II RT (200 U/µl) were added, mixed gently and incubated at 42C for 1 hour. The reaction was placed on ice, spun briefly and the second strand synthesis ingredients were added (91 µl DEPC-H2O, 30 µl 5X Second Strand Reaction Buffer, 3 µl 10 mM dNTP mix, 1 µl E.coli DNA Ligase (10 U/µl), 4 µl E.coli DNA Polymerase I (10 U/µl), 1 µl E.coli RNase H (2 U/µl)) before incubation at 16C for 2 hrs. 2 µl T4 DNA Polymerase (5 U/µl) were added and the reaction was incubated for another 5 min at 16C. The reaction was stopped by addition of 10 µl 0.5 M EDTA and phenol-chloroform extracted. The upper phase (approximately 140-150 µl was transferred to a fresh microtube and the cDNA was precipitated with 0.5 x vol 7.5 M NH4OAc and 2.5 x vol ice-cold absolute EtOH by vortexing and immediate centrifugation for 20 min at room temperature. The pellet was washed twice with ice-cold 70% EtOH and dried at 37C for 10 min before resuspending in 12 µl DEPC-treated water.

Preparation of Biotin-labelled cRNA from cDNA and fragmentation of the cDNA for hybridization Biotin-labelled cRNA was produced with the BioArray HighYield RNA Transcript Labeling Kit (ENZO) following exactly the manufacturer's instructions. 1/2 of the cDNA (6 µl) was used as starting material and the reaction was incubated in a water bath at 37C for 5 hours, gently mixing the contents of the tube every 30 min during the incubation. The labelled cRNA was purified with RNeasy Mini colums (QIAGEN) and eluted with 40 µl DEPC-treated water. 1 µg of purified and non-purified cRNA was analysed on a 1% TAE-agarose gel. The measured amount of labelled cRNA (by OD 260) was adjusted to the real amount of labelled cRNA by substracting the amount of starting total RNA (15 µg) from the measured total amount of cRNA. 15 µg of labelled cRNA (adjusted amount) per standard Affymetrix microarray were fragmented in a total volume of 30 µl by incubating with 1 x fragmentation buffer (provided as a 5 x stock: 200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc) at 94C for 30 min. 1 µg of fragmented cRNA (2 µl) were analysed by TAE-agarose gel electrophoresis.

Mice are housed under normal conditions in the animal facility of the UKE Hamburg, with the light in the room being on from 7 a.m. to 7 p.m.. They are fed with standard food from the company Ssniff (R/M Haltung)ad libitum. After killing the mice by cervical dislocation, the organs of interest were taken out, put in liquid nitrogen, and pulverized. The next step was the RNA extraction.

Transcription profiling by array of mouse Clcn5 knockout in kidney, bone and intestine

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