5 protocols
AccessionNameType
P-MTAB-27234
image_acquisition
Scanning protocol description: The scanner used was a Generation III array scanner (Molecular Dynamics/Amersham Biosciences), in association with the ArrayVision software. The oligo arrays were scanned using High Sensitivity Option. (Parameters: Scanning hardware = Generation III Array Scanner [Amersham], Scanning software = ArrayVision [Imaging Research])
P-MTAB-27236
hybridization
The hybridization was performed in the Automated Slide Processor ASP (Amersham Biosceince) with 1X Hybridization Buffer (Ambion) for 15 hours at 42C. The slides were washed 1 minute each time: once in Low Stringency Buffer (1% concentrated detergent, 5% concentrated salt – Ambion) and twice in High Stringency Buffer (0,5% concentrated salt – Ambion) (Parameters: Chamber type = OTHER: Amersham Biosceince - Automated Slide Processor ASP, Quantity of label target used = 25, Mass unit = Micro gram, time = 15, Tiny time unit = hours, Volume = 200, Volume unit = Micro litre, temperature = 42)
P-MTAB-27233
specified_biomaterial_action
In vivo electro-transfection of the thymus: Thymus electro-transfection (also referred to as electroporation) was performed based on a previously published protocol (Irla et al. 2008a,b); however, in this study, we used siRNA. Briefly, mice were anesthetized by intraperitoneal injection with a mixture of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight). A crocodile clip corresponding to the cathode was used to establish skin contact on the paw opposite to the injection site. A 0.33-mm diameter injection needle was introduced between the first and the second rib, where the thymus is located, at a 45degree angle from the longitudinal axis. We observed that inserting the needle 5 mm was necessary and sufficient to reach the thymus without affecting proximal vital organs such as the heart and lungs. After insertion of the needle, 5ul of sterile phosphate-buffered saline (PBS) or 5ul of sterile PBS containing 5 ug of Aire siRNA or irrelevant siRNA was slowly injected into each thymic lobe (5 ug siRNA per lobe, total 10ug siRNA per thymus), and an electrical current of five 20-ms pulses of 300 V was immediately applied using a standard square wave electroporator (Model ECM 830, BTX Harvard Apparatus, Holliston, MA, USA). Animals were sacrificed by CO2 inhalation 24, 48 and 72 h after electro-transfection, and thymuses were immediately removed by surgery and processed for whole thymic stroma preparation and mTEC isolation. All experiments were carried out in triplicate
P-MTAB-27235
labeling
microRNA Labeling protocol description: microRNA Labeling protocol description: Labeled microRNAs for hybridizations were generated following the protocol of mirVana miRNA Labeling Kit Ambion (cat# AM1562) . In short, the microRNAs obtained from 10ug of total RNA were dried by vacuum centrifugation. All the non-enzyme reactions components were warmed to room temperature for two hours. To the microRNAs were added: 1ul diluted Positive Control miRNA, 10ul 2X Poly(A) Polymerase reaction buffer, 2ul 25mM MnCl2, 2ul of a 10X mixture of unmodified and amine-modified nucleotides and 2ul Poly(A) Polymerase to add a nucleotide tail to each miRNA. The reaction was incubated at 37C for 2hr. To remove unincorporated nucleotides from the tailed miRNA, were added 10ul miRNA Carrier and 350ul miRNA Binding Wash Buffer, briefly mixed and incubated at room temp for 5 min. The mixture was passed through a miRNA Labeling Cartridge and centrifuged at 10000g for 15 sec. The flow-through was discarded, the miRNA Labeling Cartridge was washed with 300ul miRNA Binding Wash Buffer and centrifuged at 10000g for 15sec. The flow-through was discarded and this wash step was repeated one more time. The tailed miRNA was eluted with 2x15ul of a 95C prewarmed Elution Solution incubating at 65C for 10 min and centrifuging at 10000g for 1 min to collect the tailed miRNA in the tube each time. The samples were dried in a vacuum concentrator to post labeling with CyDye. To each dried sample were added 7ul nuclease-free water to resuspend the tailed miRNA, 9ul coupling buffer and 4ul Cy3 in DMSO. After mixing each sample was incubated for 1 hour at room temperature in the dark. To quench the reaction, 4.5 ul 4M Hydroxylamine was added and mixed and incubated for 15 minutes at room temperature in the dark. Following the protocol, 350ul miRNA Binding-Wash Buffer was added to each labeled miRNA reaction, mixed and incubated at room temperature in the dark for 5 min. The mixture passed through an miRNA Labeling Cartridge and centrifuged at 10,000g for 15 sec. The flow-through was discarded, the miRNA Labeling Cartridge was washed with 300ul miRNA Binding Wash Buffer and centrifuged at 10000g for 15sec. The flow-through was discarded and this wash step was repeated one more time. The filter was dried by centrifuging at 10,000g for 1 min. The labeled miRNA was eluted with 26ul of a 95C prewarmed Nuclease-free Water incubating at 65C for 10 min and centrifuging at 10000g for 1 min to collect the labeled miRNA in the tube. To the samples were immediately added 224ul of a 65C prewarmed 1X Hybridization Buffer. (Parameters: Amount of nucleic acid labeled = 10, Amplification = none, Mass unit = Micro gram)
P-MTAB-27237
nucleic_acid_extraction
Facility detais: 1. AROS 160 orbital shaker (Thermolyne) 2. Eppendorf Centrifugue 5415D 3. Spectrophotometer. mirVana miRNA Isolation Kit (Ambion) was used for total RNA extraction from cell cultures, according to the manufacturer's instructions. The RNA samples were eluted in H2O+DEPC and stored in a -80C freezer. The yield was quantified by spectrophotometric analysis and the integrity of RNA samples was evaluated by denaturing agarose gel electrophoresis under standard conditions, followed by Northern-blot analysis using an oligonucleotide probe recognizing the 28S rRNA fraction. (Parameters: Extracted product = total_RNA, Amplification = none)