5 protocols
Sample amplification for microarray analysis was performed using 350 ng of the isolated total RNA using Illumina Total Prep RNA Amplification Kit (Ambion, Inc., Austin, TX). The total RNA was used to produce double-stranded cDNA, followed by transcription in vitro, and by cRNA labeling with biotin-UTP. The procedure consists of reverse transcription with an oligo(dT) primer bearing a T7 promoter using a reverse transcriptase engineered to produce high yields of first strand cDNA. The reverse transcriptase catalyzes the synthesis of full-length cDNA which then undergoes second strand synthesis and clean-up to become a template for in vitro transcription (IVT) with T7 RNA Polymerase. The in vitro transcription was performed overnight (14 h) to generate single-stranded RNA molecules (cRNA). The IVT, along with biotin-16-UTP, is used to generate hundreds to thousands of biotinylated, antisense RNA copies of each mRNA.
AAV8-CMV-eGFP and AAV8-CMV-hMIF vectors were produced in SF9 insect cells by using a baculovirus based system as described elsewhere. Briefly, SF9 suspension cultures were co-infected with three baculovirus stocks: 1) Bac-Rep containing the Rep gene from AAV2, 2) Bac-Cap8 containing the Cap gene from AAV8, 3) Bac-GFP, -hMIF harboring a CMV-cDNA expression cassette (eGFP and hMIF respectively) flanked by ITRs from AAV2. Each baculovirus stock was applied with an MOI of 3. 72 hours post infection the cellular fraction was collected by centrifugation. Cell pellets were resuspended and lysed by using a French Press. For removal of genomic DNA cell lysates were incubated with benzonase (50 U/mL) for 1 hour at 37C. Subsequently AAV particles were precipitated with CaCl2 (25 mM) followed by PEG precipitation (8% PEG-8000, 500 mM NaCl). After resuspension of PEG precipitates in 50 mM HEPES, 150 mM NaCl, 25 mM EDTA, pH 7.4 over night at 4C, AAV particles were further purified by CsCl density gradient centrifugation. Fractions from CsCl density gradients were analyzed by measuring the refractory index (RI) using a digital refractometer. Samples with a RI between 1.3774 and 1.3696 were pooled and dialyzed against PBS for removal of CsCl with a molecular weight cutoff of 20 kDa. Finally AAV preparations were further concentrated by using ultra filtration units. After addition of glycerol to a final concentration of 10%, AAV preparations were sterile filtered, aliquoted, frozen and subsequently stored at -80C.Genomic titers of purified AAV stocks were determined by isolation of vector-DNA and subsequent qPCR analysis using CMV-promoter specific primers.
Tissue samples were treated prior to the isolation of nucleic acids with a phenol-chloroform extraction protocol. First, the samples were homogenized in Lysing Matrix D Tubes containing 700 uL RLT-buffer (Rneasy Lysis Buffer, 2-Mercaptoethanol added) with the FastPrep-24 Instrument from Biomedicals (3000×g, 3 min). The supernatants were transferred and 700 uL phenol-chloroform-isoamyl alcohol were added. Tubes were mixed and centrifuged at 12000×g for 5 min and then 500 uL chloroform-isoamyl alcohol were added and incubated for 3 min at room temperature. After another centrifugation step, the top phase was used for the extraction of RNA. Total RNA was isolated according to the Rneasy protocol from Qiagen (Cat#74182). RNA concentrations were analyzed by NanoDrop ND-1000 UV-Vis Spectrophotometer at 260 nm (Thermo Scientific). RNA purity was assessed with the 260/280 value (pure samples show a range of 1.8 – 2.1) and samples were stored at -80C.
C57BL/6 or Balb/c mice were obtained from Janvier (France). In single experiments gender and sex matched animals were used. All animal studies were performed in accordance with the German Law on the Protection of Animals