Return to study ST002416 main page
MB Sample ID: SA242053
Local Sample ID: | N03-01_9_1_1407 |
Subject ID: | SU002505 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002505 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
N03-01_9_1_1407 | SA242053 | FL030336 | Normal | Group |
Collection:
Collection ID: | CO002498 |
Collection Summary: | The experiments on the mice were approved by the University of Sharjah animal care and use committee (ACUC) (ACUC approval number: ACUC-16-003-EAE and ACUC-21-09-06-01). The mice were housed at the University of Sharjah animal facility, in plastic cages under a controlled environment of 24 ± 2 °C, 50% ± 5% humidity, 12 h/12 h light-dark cycle with unlimited food and water access. |
Sample Type: | Brain |
Treatment:
Treatment ID: | TR002517 |
Treatment Summary: | From Normal, EAE vehicle-treated, and therapeutic SIMR1707 treated mice, the cerebellum part of the brain was cut, and immediately snap frozen using liquid nitrogen. The samples then were weighed, made in powder form and homogenized using pistol method under liquid nitrogen. The cerebellum sample was lysed using 400 uL RIBA lysis buffer and let to set for 5-10 minutes to allow lysis. 50 ul of the homogenized sample was taken for RNA extraction using norgen biotek RNA/DNA purification kit (cat#:47700) according to the manufacturer’s instructions. The remaining 350 uL was processed for metabolites and proteins extraction. First, it was centrifuged for 5 minutes at 4 C, 13000 rpm. The supernatant was collected in a new 1.5 mL eppendorf tube. The supernatant was centrifuged again for 1 minute at 4 C, at maximum speed (>13000 rpm), and the supernatant was collected again in a new 1.5 mL tube and kept on ice as it was used in the following steps, while the pellet was discarded. |
Sample Preparation:
Sampleprep ID: | SP002511 |
Sampleprep Summary: | 400 uL of methanol, and 300 uL of chloroform were added to the protein sample, and centrifuged for 5 minutes, at 4 C, 13000 rpm. Three phases appeared: the upper phase is the metabolites, the middle is the protein, and the lower phase is other metabolites. The metabolites' upper phase was kept in a new eppendorf tube and kept at -20 C. 300 uL methanol was added to the middle and the lower phases then they were mixed and centrifuged for 1 minute at 13000 rpm. The supernatant was added to the metabolite tube and then it was kept at - 80C for longer storage. |
Combined analysis:
Analysis ID | AN003937 |
---|---|
Analysis type | MS |
Chromatography type | Reversed phase |
Chromatography system | Bruker Elute |
Column | Hamilton Intensity Solo 2 C18(100 x 2.1 mm, 1.8 um) |
MS Type | ESI |
MS instrument type | QTOF |
MS instrument name | Bruker timsTOF |
Ion Mode | POSITIVE |
Units | AU |
Chromatography:
Chromatography ID: | CH002916 |
Instrument Name: | Bruker Elute |
Column Name: | Hamilton Intensity Solo 2 C18(100 x 2.1 mm, 1.8 um) |
Column Temperature: | 35 |
Flow Gradient: | 1%B to 99%B in 15 min |
Flow Rate: | 250 uL/min |
Solvent A: | 100% water; 0.1% formic acid |
Solvent B: | 100% acetonitrile; 0.1% formic acid |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS003675 |
Analysis ID: | AN003937 |
Instrument Name: | Bruker timsTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | Metabolomics Data Processing. For metabolomic analysis, MetaboScape® 4.0 program (Bruker Daltonics, Billerica, MA, USA) was employed for data processing, feature extrac-tion and metabolite identification. The T-ReX 2D/3D workflow was used to identify the molecular features with the following settings: The minimum peak length was set to 7 spectra and the minimum intensity threshold was 1000 counts for peaks detection. The peak area was employed for quantification and the injected external calibrant in the in-terval of 0–0.3 min was used to recalibrate the mass spectra. The selected mass to charge ratio (m/z) and retention time for scanning were in the ranges of 20–1300 m/z and 0.3–30 min, respectively. MS/MS spectra for features were averaged on import and features found at least in 12 of the 40 injections were taken into further consideration. Metabolites were identified by matching to the human metabolome database (HMDB) by combined MS/MS, precursor m/z values, and isotopic pattern scores. Where multiple features matched a given database entry, the annotation quality score (AQ score) was used to select only the best matching feature. |
Ion Mode: | POSITIVE |