Return to study ST001666 main page
MB Sample ID: SA152231
Local Sample ID: | 26 |
Subject ID: | SU001743 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Age Or Age Range: | 8 weeks |
Gender: | Male |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU001743 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Age Or Age Range: | 8 weeks |
Gender: | Male |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
26 | SA152231 | FL017904 | GFP | Treatment |
Collection:
Collection ID: | CO001736 |
Collection Summary: | Fed male C57BL/6J mice were anesthetized with 5% isoflurane, and isolated hearts were perfused in the Langendorff mode at 37°C with non-recirculating perfusate. The hearts were allowed to beat spontaneously throughout the perfusion. At the end of each perfusion, hearts were immediately freeze-clamped in liquid nitrogen using the Wollenberger technique and stored at -80 oC for further analysis. |
Sample Type: | Heart |
Treatment:
Treatment ID: | TR001756 |
Treatment Summary: | SLC25A44 and GFP expression plasmids with a CMV promoter containing ITRs were used to prepare recombinant AAV9 by the UMass Gene Therapy Core. Adult 8-week-old male mice were injected intravenously with 100 µl of 5.1 x 1011 vector genomes per mouse. |
Sample Preparation:
Sampleprep ID: | SP001749 |
Sampleprep Summary: | Frozen tissues were pulverized under liquid nitrogen using a mortar and pestle. Metabolites were then extracted using sequential 500 μL additions of -20°C MeOH, chilled water, and chloroform. After each addition, tissue lysates were prepared with a Tissue Lyser (Qiagen) for 60 seconds at 30Hz. Similarly, plasma metabolites (20 μL) were extracted by sequential 500 μL additions of -20°C MeOH, chilled water, and chloroform. After each addition, samples were vortexed for 30 seconds. Tissue extracts were then centrifuged at 4°C and 14400 x g for 20 minutes and the clarified aqueous phase was transferred to a fresh Eppendorf and stored in -80°C until processing for GC-MS analysis. For GC-MS analysis, the extracted tissue was dried under N2 gas-flow at 37°C using an evaporator. Amino and organic acids were derivatized via methoximation and silylation. Briefly, metabolites were resuspended in 25 μL of methoxylamine hydrochloride (2% (w/v) in pyridine) and incubated at 40°C for 90 minutes on a heating block. After brief centrifugation, 35 μL of MTBSTFA + 1% TBDMS was added and the samples were incubated at 60°C for 30 minutes. |
Combined analysis:
Analysis ID | AN002718 |
---|---|
Analysis type | MS |
Chromatography type | GC |
Chromatography system | Agilent 7890B |
Column | Agilent HP5-MS (30m x 0.25mm, 0.25 um) |
MS Type | EI |
MS instrument type | Single quadrupole |
MS instrument name | Agilent 5977A |
Ion Mode | UNSPECIFIED |
Units | Concentration of 13C (uM) |
Chromatography:
Chromatography ID: | CH002006 |
Instrument Name: | Agilent 7890B |
Column Name: | Agilent HP5-MS (30m x 0.25mm, 0.25 um) |
Chromatography Type: | GC |
MS:
MS ID: | MS002515 |
Analysis ID: | AN002718 |
Instrument Name: | Agilent 5977A |
Instrument Type: | Single quadrupole |
MS Type: | EI |
MS Comments: | Masshunter used for data acquisition and processing. |
Ion Mode: | UNSPECIFIED |