Metabolomic analysis was performed using gas chromatography/mass spectrometry (GC/MS) as previously described [18 (link)], with some modifications. Approximately 20 mg of the freeze-dried muscle samples or 50 μL of plasma was suspended in 250 µL of methanol–chloroform–water (5:2:2) and 5 µL of 1 mg/mL 2-isopropylmalic acid as an internal standard. The samples were then mixed in a shaker at 1200 rpm at 37 °C for 30 min and then centrifuged at 16,000× g at 4 °C for 5 min. Next, 225 µL of the supernatant was mixed with 200 µL of distilled water and vortexed, followed by centrifugation at 16,000× g at 4 °C for 5 min. Subsequently, 250 µL of the supernatant was dried under vacuum using a centrifugal evaporator (RD-400; Yamato Scientific, Tokyo, Japan) after cooling at −80 °C for 10 min. Methoxyamine hydrochloride in pyridine (20 mg/mL, 40 µL) was then added to the tubes, vortex-mixed, and shaken at 1200× g at 30 °C for 90 min in the dark for oximation. N-methyl-N-trimethylsilyltrifluoroacetamine (20 µL) was then added to each tube, and the contents were vortex-mixed. Trimethylsilyl derivatives were prepared by shaking the tubes at 1200× g at 37 °C for 45 min in the dark.
Rd 400
Manufactured by Yamato Scientific
Sourced in Japan
The RD-400 is a laboratory instrument designed for sample preparation and analysis. It features a high-precision rotational speed control system and a durable, corrosion-resistant construction. The RD-400 is suitable for a variety of applications requiring controlled mixing or agitation of samples.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using rd 400
1
Metabolomic Analysis of Muscle and Plasma
2
GC-MS Metabolomic Analysis of Plasma
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Metabolomic analysis was performed using GC-MS
as previously described[12 (link)], with some modifications.
Fifty microliters of plasma was suspended in 250
µL of methanol:chloroform:water (5:2:2) and 5 µL
of 1 mg/mL 2-isopropylmalic acid as an internal
standard. Samples were mixed in a shaker at 1,200
rpm at 37°C for 30 min and then centrifuged at
16,000 ×g at 4°C for 5 min. Next,
225 µL of the supernatant was vortex-mixed with
200 µL of distilled water, followed by
centrifugation at 16,000 ×g at
4°C for 5 min. Subsequently, 250 µL of the
supernatant was dried under a vacuum using a
centrifugal evaporator (RD-400; Yamato Scientific,
Tokyo, Japan) after cooling at –80 °C for 10 min.
Methoxyamine hydrochloride in pyridine (20 mg/mL,
40 µL) was then added to the tubes, which were
vortex-mixed and then shaken at 1,200
×g at 30°C for 90 min in the dark
for oximation.
N-methyl-N-trimethylsilyltrifluoroacetamine (20
µL) was then added to each tube and the contents
were vortex-mixed. To prepare trimethylsilyl
derivatives, the tubes were shaken at 1,200
×g at 37°C for 45 min in the
dark.
as previously described[12 (link)], with some modifications.
Fifty microliters of plasma was suspended in 250
µL of methanol:chloroform:water (5:2:2) and 5 µL
of 1 mg/mL 2-isopropylmalic acid as an internal
standard. Samples were mixed in a shaker at 1,200
rpm at 37°C for 30 min and then centrifuged at
16,000 ×g at 4°C for 5 min. Next,
225 µL of the supernatant was vortex-mixed with
200 µL of distilled water, followed by
centrifugation at 16,000 ×g at
4°C for 5 min. Subsequently, 250 µL of the
supernatant was dried under a vacuum using a
centrifugal evaporator (RD-400; Yamato Scientific,
Tokyo, Japan) after cooling at –80 °C for 10 min.
Methoxyamine hydrochloride in pyridine (20 mg/mL,
40 µL) was then added to the tubes, which were
vortex-mixed and then shaken at 1,200
×g at 30°C for 90 min in the dark
for oximation.
N-methyl-N-trimethylsilyltrifluoroacetamine (20
µL) was then added to each tube and the contents
were vortex-mixed. To prepare trimethylsilyl
derivatives, the tubes were shaken at 1,200
×g at 37°C for 45 min in the
dark.
3
GC/MS Liver Metabolomics Protocol
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A liver metabolomic analysis was performed using GC/MS as described previously (15) with some modifications. In brief, freeze-fractured liver samples (approximately 20 mg) were suspended in 250 μl of methanol-chloroform-water (2•5:1:1) and 5 μl of 1 mg/ml 2-isopropylmalic acid as an internal standard and homogenised using a polytron homogeniser (Microtec; China). Samples were subsequently mixed in a shaker at 1200 rpm at 37°C for 30 min and centrifuged at 16 000 g at 4°C for 5 min. Next, 160 μl of the supernatant was mixed with 200 μl of distilled water and vortexed, followed by centrifugation at 16 000 g at 4°C for 5 min; subsequently, 250 μl of the supernatant was dried under a vacuum using a centrifugal evaporator (RD-400; Yamato Scientific). Dried samples were pre-treated, derivatised and analysed by GC/MS (GCMS QP2010-Ultra; Shimadzu) within 24 h of derivatisation as described by Qiao et al. (15) . The Shimadzu Smart Metabolites Database (Shimadzu) was used to identify metabolites. The DNA content in the liver was analysed using the method by Labarca & Paigen (16) . The relative metabolite content was calculated as follows: the peak area of each metabolite was divided by that of the internal standard, 2-isopropylmalic acid, on the same chromatogram. It was further divided by the DNA content. The level for each metabolite in the control liver was set at 100.
4
Plasma Metabolite Extraction and Derivatization
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Fifty μL aliquots of plasma were suspended in 250 µL of methanol/chloroform/water (5:2:2), with 5 µL of 1 mg/mL 2-isopropylmalic acid as the internal standard. The samples were then mixed in a shaker at 1200 rpm at 37 °C for 30 min, and then centrifuged at 16,000× g at 4 °C for 5 min. Next, 225 µL of the supernatant was mixed with 200 µL of distilled water and vortex-mixed, followed by centrifugation at 16,000× g at 4 °C for 5 min. Subsequently, 250 µL of the supernatant was dried under a vacuum using a centrifugal evaporator (RD-400; Yamato Scientific, Tokyo, Japan), after cooling at −80 °C for 10 min. Methoxyamine hydrochloride in pyridine (20 mg/mL, 40 µL) was then added to the tubes, and they were vortex-mixed, then shaken at 1200× g at 30 °C for 90 min in the dark to allow oximation. N-methyl-N-trimethylsilyltrifluoroacetamine (20 µL) was then added to each tube, and the contents were vortex-mixed. To prepare trimethylsilyl derivatives, the tubes were shaken at 1200× g at 37 °C for 45 min in the dark.
5
GC/MS Metabolomic Analysis of Liver and Plasma
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Metabolomic analysis was performed using gas chromatography/mass spectrometry (GC/MS) as previously described [20 (link)], with some modifications. Frozen liver samples were pulverized into a powder using a crusher (T- 351, Tokyo Unicom, Tokyo, Japan) with frozen carbon dioxide. Approximately 20 mg of the freeze-fractured liver samples and cecal content samples and 50 μL of plasma were then suspended in 250 µL of methanol–chloroform–water (5:2:2) and 5 µL of 1 mg/mL 2-isopropylmalic acid as an internal standard. Samples were then mixed in a shaker at 1200 rpm at 37 °C for 30 min, then centrifuged at 16,000× g at 4 °C for 5 min. Next, 225 µL of the supernatant was mixed with 200 µL of distilled water and vortex-mixed, followed by centrifugation at 16,000× g at 4 °C for 5 min. Subsequently, 250 µL of the supernatant was dried under a vacuum using a centrifugal evaporator (RD-400; Yamato Scientific, Tokyo, Japan) after cooling at −80 °C for 10 min. Methoxyamine hydrochloride in pyridine (20 mg/mL, 40 µL) was then added to the tubes and they were vortex-mixed, then shaken at 1200× g at 30 °C for 90 min in the dark for oximation. N-methyl-N-trimethylsilyltrifluoroacetamine (20 µL) was then added to each tube and the contents were vortex-mixed. To prepare trimethylsilyl derivatives, the tubes were shaken at 1200× g at 37 °C for 45 min in the dark.
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