Summary of Study ST003658
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002268. The data can be accessed directly via it's Project DOI: 10.21228/M8G54G This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
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| Study ID | ST003658 |
| Study Title | Functional analysis of retinal pigment epithelial cells with PNPLA6 knockdown |
| Study Summary | PNPLA6 was knocked down using siRNA on ARPE-19, a representative cultured cell line of human retinal pigment epithelial cells. Lipid analysis of the constituent phospholipids was performed on these cells, comparing them to a group treated with scramble siRNA. We also overexpressed PNPLA6 in human retinal pigment epithelial cells ARPE-19 and performed the same lipid analysis. |
| Institute | University of Tokyo |
| Last Name | Ono |
| First Name | Takashi |
| Address | 7-3-1, Hongo, Bunkyo-ku |
| taono-tky@umin.ac.jp | |
| Phone | 0338155411 |
| Submit Date | 2024-12-01 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | API |
| Release Date | 2025-01-26 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002268 |
| Project DOI: | doi: 10.21228/M8G54G |
| Project Title: | Role of lipid-metabolizing enzyme PNPLA6 in retinal pigment epithelial cells and its mechanism of homeostasis |
| Project Summary: | PNPLA6 deficiency in the retina has been clinically reported to be a cause of retinitis pigmentosa, a disease leading to blindness. PNPLA6 is generally known to be an enzyme that degrades phospholipids in a calcium-independent manner, but its activity and substrates in the eye have not yet been elucidated. We focused on the function of PNPLA6, whose function in the retina is not well understood, and biochemically analyzed the mechanism by which retinal pigmentary degeneration occurs. |
| Institute: | University of Tokyo |
| Last Name: | Ono |
| First Name: | Takashi |
| Address: | 7-3-1, Hongo, Bunkyo-ku |
| Email: | taono-tky@umin.ac.jp |
| Phone: | +81-338155411 |
Subject:
| Subject ID: | SU003788 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Genotype |
|---|---|---|---|
| SA398941 | PN6KD-1 | RPE (Retinal pigment epithelium) | KD |
| SA398942 | PN6KD-2 | RPE (Retinal pigment epithelium) | KD |
| SA398943 | PN6KD-3 | RPE (Retinal pigment epithelium) | KD |
| SA398944 | PN6KD-4 | RPE (Retinal pigment epithelium) | KD |
| SA398953 | mock-3 | RPE (Retinal pigment epithelium) | mock |
| SA398954 | mock-4 | RPE (Retinal pigment epithelium) | mock |
| SA398955 | mock-2 | RPE (Retinal pigment epithelium) | mock |
| SA398956 | mock-1 | RPE (Retinal pigment epithelium) | mock |
| SA398945 | PN6OE-1 | RPE (Retinal pigment epithelium) | OE |
| SA398946 | PN6OE-4 | RPE (Retinal pigment epithelium) | OE |
| SA398947 | PN6OE-3 | RPE (Retinal pigment epithelium) | OE |
| SA398948 | PN6OE-2 | RPE (Retinal pigment epithelium) | OE |
| SA398949 | NegKD-1 | RPE (Retinal pigment epithelium) | Wild |
| SA398950 | NegKD-2 | RPE (Retinal pigment epithelium) | Wild |
| SA398951 | NegKD-4 | RPE (Retinal pigment epithelium) | Wild |
| SA398952 | NegKD-3 | RPE (Retinal pigment epithelium) | Wild |
| Showing results 1 to 16 of 16 |
Collection:
| Collection ID: | CO003781 |
| Collection Summary: | The human RPE cell line ARPE-19 (CRL-2302, ATCC) was cultured in Dulbecco's Modified Eagle's Medium (DMEM)/F12 and DMEM high glucose , respectively, with penicillin streptomycin (100 units/ml) and 10% (v/v) fetal bovine serum in a CO2 incubator under 5% (v/v) CO2 at 37°C. Target-specific Silencer® Select siRNAs (Thermo Fischer Scientific) or a Silencer® Select Negative Control No. 1 siRNA (4390843, Thermo Fischer Scientific) were transfected into ARPE-19 with Lipofectamine 3000. Cells were collected once after transfection, re-spread, and collected 3 days later. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003797 |
| Treatment Summary: | Cells were stripped using trypsin EDTA, washed with PBS, and cell counts were performed with automated cell counter. |
Sample Preparation:
| Sampleprep ID: | SP003795 |
| Sampleprep Summary: | Total lipids were extracted using the Bligh and Dyer method. After the samples were dried, 175 µL of digestion reagent (60% HClO: H2SO4 = 1:1 (v/v)) was added to the samples or a phosphorus standard (NaH2PO4), stirred, and scorched using a gas burner. After cooling at room temperature, 125 µL of water, 1 mL of 1% (w/v) ammonium molybdate, and 50 µL of reducing reagent were added and the samples were boiled for 10 min. The reaction was stopped on ice, and the absorbance was measured at 750 nm. |
Combined analysis:
| Analysis ID | AN006008 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | LC-30AD |
| Column | SeQuant ZIC-HILIC (100 x 2.1 mm, 3.5 µm) |
| MS Type | API |
| MS instrument type | QTRAP |
| MS instrument name | ABI Sciex 4000 QTrap |
| Ion Mode | POSITIVE |
| Units | nmol/10^6 cells |
Chromatography:
| Chromatography ID: | CH004565 |
| Instrument Name: | LC-30AD |
| Column Name: | SeQuant ZIC-HILIC (100 x 2.1 mm, 3.5 µm) |
| Column Temperature: | 50°C |
| Flow Gradient: | 0–3 min 0% B; 3–6 min 0–60% B; 6–17 min 60–62.2% B; 17–17.01 min 62.2–69.8% B; 17.01–20 min 69.8–100% B; 20–23 min 100% B; 23–23.10 min 100–0% B; 23.10–26 min 0% B |
| Flow Rate: | 0.2 mL/min |
| Solvent A: | 95% Acetonitrile/5% Water; 10 mM ammonium acetate |
| Solvent B: | 50% Acetonitrile/50% Water; 20 mM ammonium acetate |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS005719 |
| Analysis ID: | AN006008 |
| Instrument Name: | ABI Sciex 4000 QTrap |
| Instrument Type: | QTRAP |
| MS Type: | API |
| MS Comments: | 4500 QTRAP LC-MS/MS/MS system (AB Sciex), a triple quadrupole lipid molecular apparatus, was employed to quantify individual lipid molecules. Lipid samples (5 nmol of phospholipids) or standard lipids (Avanti Polar Lipids), prepared as previously described, were transferred to vials with added internal standards for each lipid type. The solvent was then evaporated using nitrogen gas, and the residue was dissolved in 50 µL of isopropanol:methanol (1:1, v/v) before being placed in an autosampler. Lipid profiling was conducted using a concurrent quantification method for phospholipids and lysophospholipids via multiple reaction monitoring (MRM). MultiQuant software (AB Sciex) was utilized to analyze the data, yielding peak area under the curve (AUC) values for each lipid molecule. Quantification of individual lipid species was achieved using calibration curves generated from AUCs obtained at LC elution times matching those of the standard lipids. |
| Ion Mode: | POSITIVE |
