Lipid biomarker for autism and application of lipid biomarker
A biomarker and autism technology, applied in the field of disease diagnosis, can solve the problems of few literature reports
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Embodiment 1
[0022] Example 1 16p11.2 + / - Microstructural changes of myelin sheath in mouse striatum
[0023] Mice were anesthetized with 0.1 g / ml chloral hydrate and the heart was perfused first with 20 ml of 1X Phosphate Buffered Saline (PBS) followed by 60 ml of fixative (4% PFA + 2.5% glutaraldehyde in 1X PBS) . Brains were removed and striatum were harvested using brain molds, which were then transferred to EP tubes filled with fresh TEM fixative (Servicebio) for further fixation, stored and shipped at 4°C.
[0024] Tissues were washed 3 times with 0.1 M PB (pH 7.4) for 15 minutes each, and then fixed in 1% OsO4 in 0.1 M PB (pH 7.4) for 2 hours at room temperature protected from light. After removal of OsO4, the tissue was rinsed three times for 15 minutes each in 0.1 M PB (pH 7.4). Dehydrate the tissue at room temperature as follows: 30% ethanol for 20 min; 50% ethanol for 20 min; 70% ethanol for 20 min; 80% ethanol for 20 min; 95% ethanol for 20 min; two changes of 100% ethanol f...
Embodiment 2
[0027] Example 2 Myelin-related genes at 16p11.2 + / - Expression changes in mouse striatum
[0028] (1) RNA sequencing
[0029] Four wild-type (WT) female mice and four 16p11.2 mice were used at postnatal day 60 + / - female mice. The mice were terminally anesthetized with 0.1 g / ml chloral hydrate (Macklin, Shanghai), and the striatal tissue was excised and immediately frozen in liquid nitrogen. RNA extraction and RNA sequencing (RNAseq) analysis were performed by Applied Protein Technology Co. Briefly, total RNA was isolated using TRIzol RNA Isolation Reagent and treated with RNase-free DNase I to remove genomic DNA. A total of 2 μg RNA per sample was used as input material for RNA sample preparation. A cDNA library (paired-end 250bp, PE250) was constructed, and HiSeq 2000 sequencing system was used (high-throughput sequencing was performed). After filtering out low-quality sequences and adapter sequences, reads were mapped to the mouse genome using Hisat2 software (versio...
Embodiment 3
[0035] Example 3 16p11.2 + / - Altered lipidomic profiles in the mouse striatum
[0036] Six WT female mice and five 16p11.2 mice were used at P60 + / - female mice. Mice were terminally anesthetized with 0.1 g / ml chloral hydrate, striatal tissue was excised and immediately frozen in liquid nitrogen. Lipid extraction and mass spectrometry-based lipid detection were performed by Applied Protein Technology Co. A small fraction of samples from each group (WT and 16p11.2 + / - ) to create pooled QC samples. Insert quality control samples into the analysis queue to assess system stability and data reliability throughout the experiment. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) was performed on a Q Exactive plus mass spectrometer (Thermo Fisher Scientific) combined with UHPLCNexera LC-30A (Shimadzu Co. Beijing). Lipid identification, peak extraction, peak alignment and quantification were evaluated using LipidSearch software version 4.1 (Thermo Fisher Scientific).
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