Affymetrix GeneChip Murine Genome U74Av2 [MG_U74Av2]

Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6

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3 pairs of wt and ClC-6 knockout mice, RNA from p14 hippocampus

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Transcription profiling by array of hippocampus from CIC-6 knock-out mice

Transcription profiling of hippocampus from wild type and CIC-6 knock-out mice

3 pairs of wt and ClC-6 knockout mice, RNA from p14 hippocampus


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:

P-MEXP-6737

Preparation of total RNA from ClC7 wild-type and knock-out hippocampus. For the expression analysis, we used three pairs of mice: P14 wild-type and ClC7 knock-out litter mates, which were killed at the same time (9.30 am) by cervical dislocation. The complete brain of these mice was removed from the skull and put into a petri-dish containing sterile PBS. The two hippocampal halves were dissected from these brains by performing a sagittal cut and preparing the hippocampus from each brain half under a microscope. The brain tissue remained in PBS during the whole dissection procedure. The hippocampus was then immediately frozen on an aluminium foil-wrapped metal plate on dry ice, transferred to a pre-cooled microtube, frozen in liquid nitrogen and stored at -80°C until RNA preparation.

P-MEXP-6738

Total RNA was prepared from one hippocampal half of each mouse by immediate transfer of the frozen tissue into 800 µl TRIZOL (Gibco BRL). The tissue was disrupted with 2 ml syringes (10 x 0.90 x 40 mm needle, then 10 x 0.45 x 25 mm needle). The homogenized tissue was incubated in TRIZOL at room temperature for 5 min before addition of 160 µl (0.2 x vol TRIZOL) chloroform. The mixture was shaken vigorously for 15 sec and incubated at room temperature for 3 min before centrifugation for 10 min at 4°C with 10000 rpm. The upper phase (0.4 ml) was transferred carefully into a fresh microtube and 0.4 ml isopropanol was added per tube. After vortexing, the tube was incubated for 10 min at room temperature before centrifugation at 14000 rpm, 4°C, 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 70°C 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 42°C for 2 min to equilibrate the temperature. 2 µl SuperScriptTM II RT (200 U/µl) were added, mixed gently and incubated at 42°C 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 16°C for 2 hrs. 2 µl T4 DNA Polymerase (5 U/µl) were added and the reaction was incubated for another 5 min at 16°C. 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 37°C for 10 min before resuspending in 12 µl DEPC-treated water.

P-MEXP-6739

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 37°C 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 94°C for 30 min. 1 µg of fragmented cRNA (2 µl) were analysed by TAE-agarose gel electrophoresis.

Transcription profiling by array of hippocampus from CIC-6 knock-out mice

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