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MB Sample ID: SA163301

Local Sample ID:NYSM-00454
Subject ID:SU001822
Subject Type:Mammal
Subject Species:Rattus norvegicus
Taxonomy ID:10116
Genotype Strain:Long Evans
Age Or Age Range:From post-natal day 1 (PN1) to PN80
Weight Or Weight Range:5g-250g
Gender:Male and female
Animal Animal Supplier:Envigo
Animal Housing:Animal facility at NYU
Animal Light Cycle:7am-7pm

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Subject:

Subject ID:SU001822
Subject Type:Mammal
Subject Species:Rattus norvegicus
Taxonomy ID:10116
Genotype Strain:Long Evans
Age Or Age Range:From post-natal day 1 (PN1) to PN80
Weight Or Weight Range:5g-250g
Gender:Male and female
Animal Animal Supplier:Envigo
Animal Housing:Animal facility at NYU
Animal Light Cycle:7am-7pm

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
NYSM-00454SA163301FL019446naiveTreatment

Collection:

Collection ID:CO001815
Collection Summary:Rats trained in inhibitory avoidance at PN17, PN24, and PN80, and the untrained (naive) control rats were euthanized by decapitation (1 hour after IA training). Their brains were quickly removed and placed in ice-cold phosphate-buffered saline (1X). Whole hippocampal samples were dissected and immediately snap-frozen in isopentane on dry ice. All samples were stored at -80°C until processing. Frozen samples were shipped in dry ice to Metabolon Inc. (Morrisville, NC, USA) for metabolomic analysis using a proprietary methodology. Extraction of samples was performed using a MicroLab STAR automated liquid handling robot (Hamilton Robotics, Inc., Reno, NV, USA); 450 μL of methanol was added to 100 μo of sample to precipitate proteins. Four recovery standards (DL-2-fluorophenylglycine, tridecanoic acid, cholesterol-d6 and 4-chlorophenylalanine) were added to each sample to determine extraction efficiency. To remove proteins, dissociate small molecules bound to protein, and recover metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation (1300 g for 10 min at room temperature). The resultant supernatants were placed in a TurboVap (Zymark) to remove the organic solvent. Each sample extract was then divided into five equal aliquots, dried under nitrogen, and stored in vacuo prior to metabolomic profiling.
Sample Type:Brain
Collection Method:Brain dissection
Collection Location:New York University
Storage Conditions:-80℃

Treatment:

Treatment ID:TR001835
Treatment Summary:Metabolomic profiling was carried out on hippocampal extracts (five to seven individual replicates) obtained from untrained rats at four early, prepuberal developmental ages [postnatal day 1 (PN1), PN7, PN17 and PN24] and compared to the profile obtained from young adults (PN80). Additionally, the metabolomic profiles of the hippocampus 1 hour after IA training at PN17, PN24, and PN80 were compared with those of age-matched untrained (naive) control rats.

Sample Preparation:

Sampleprep ID:SP001828
Sampleprep Summary:Extraction of samples was performed using a MicroLab STAR automated liquid handling robot (Hamilton Robotics, Inc., Reno, NV, USA); 450 μL of methanol was added to 100 μL of sample to precipitate proteins. Four recovery standards (DL-2-fluorophenylglycine, tridecanoic acid, cholesterol-d6 and 4-chlorophenylalanine) were added to each sample to determine extraction efficiency. To remove proteins, dissociate small molecules bound to protein, and recover metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation (1300 g for 10 min at room temperature). The resultant supernatants were placed in a TurboVap (Zymark) to remove the organic solvent. Each sample extract was then divided into five equal aliquots, dried under nitrogen, and stored in vacuo prior to metabolomic profiling.

Combined analysis:

Analysis ID AN002838 AN002839 AN002840 AN002841
Analysis type MS MS MS MS
Chromatography type Reversed phase Reversed phase Reversed phase HILIC
Chromatography system Waters Acquity Waters Acquity Waters Acquity Waters Acquity
Column Waters Acquity BEH C18 (100 x 2mm, 1.7um) Waters Acquity BEH C18 (100 x 2mm, 1.7um) Waters Acquity BEH C18 (100 x 2mm, 1.7um) Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
MS Type ESI ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE POSITIVE NEGATIVE NEGATIVE
Units pmoles/l pmoles/l pmoles/l pmoles/l

Chromatography:

Chromatography ID:CH002101
Chromatography Summary:The first two aliquots were analyzed by two separate reverse-phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI): one aliquot was analyzed using acidic positive-ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient-eluted from a C18 column (2 mm × 100 mm Waters UPLC BEH C18 1.7 µm column held at 40°C) using 5% water and 95% methanol and containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA) at pH 3.5. The second aliquot was also analyzed using acidic positive-ion conditions; however, it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient-eluted from the aforementioned C18 column using methanol, acetonitrile, water, 0.05% PFPA, and 0.01% FA at pH 3.5. A third aliquot was analyzed by RP/UPLC-MS/MS using basic negative-ion mode ESI in a separate dedicated C18 column. The basic extracts were gradient-eluted from the column using methanol (95%) and water (5%) with 6.5 mM ammonium bicarbonate at pH 8.
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (100 x 2mm, 1.7um)
Chromatography Type:Reversed phase
  
Chromatography ID:CH002102
Chromatography Summary:The fourth aliquot was analyzed via negative ionization following elution from a Hydrophilic-Interaction Chromatography (HILIC) column (2.1 mm x 150 mm Waters UPLC BEH Amide, 1.7 µm column held at 40°C) using a mobile phase consisting of 10 mM ammonium formate in 15% water, 5% methanol, 80% acetonitrile (pH 10.8).
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
Chromatography Type:HILIC

MS:

MS ID:MS002631
Analysis ID:AN002838
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:One aliquot was analyzed using acidic positive-ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient-eluted from a C18 column (2.1 mm × 100 mm Waters UPLC BEH C18 1.7 µm column held at 40°C) using 5% water and 95% methanol and containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA) at pH 3.5.
Ion Mode:POSITIVE
  
MS ID:MS002632
Analysis ID:AN002839
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The second aliquot was also analyzed using acidic positive-ion conditions; however, it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient-eluted from the aforementioned C18 column using methanol, acetonitrile, water, 0.05% PFPA, and 0.01% FA at pH 3.5.
Ion Mode:POSITIVE
  
MS ID:MS002633
Analysis ID:AN002840
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:A third aliquot was analyzed by RP/UPLC-MS/MS using basic negative-ion mode ESI in a separate dedicated C18 column. The basic extracts were gradient-eluted from the column using methanol (95%) and water (5%) with 6.5 mM ammonium bicarbonate at pH 8.
Ion Mode:NEGATIVE
  
MS ID:MS002634
Analysis ID:AN002841
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The fourth aliquot was analyzed via negative ionization following elution from a Hydrophilic-Interaction Chromatography (HILIC) column (2.1 mm x 150 mm Waters UPLC BEH Amide, 1.7 µm column held at 40°C) using a mobile phase consisting of 10 mM ammonium formate in 15% water, 5% methanol, 80% acetonitrile (pH 10.8).
Ion Mode:NEGATIVE
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