biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsKyonoshin Maruyamatranscription profiling by arrayOryza sativaOryza sativahttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15013We conducted an oligo microarray analysis to investigate the gene transcripts in the roots of NB and DJ123 grown in LS and control hydroponic conditionsbiostudies-arrayexpressNucleic Acid Extraction - After N2 evaporates, add 1 m1 of RNAiso Plus to each sample tube and mix well using micro tube mixer for 5-10 minutes. Centrifuge at 12,000 g for 15 minutes at 4°C and transfer 800 μl of supernatant to a new tube. Add chloroform (200-400 μl) to each sample tube and mix well using micro tube mixer for 5 minutes at room temperature. Centrifuge at 12,000 g for 10 minutes at 4°C and transfer 400 μl the top layer to a new tube. Add 250 μl of high salt buffer and 250 μl of isopropanol to each sample tube and mix well using micro tube mixer for 5 minutes at room temperature. Centrifuge at 12,000 g for 10 minutes at 4°C and after removal of supernatant carefully, dissolve in 100 μl of ultrapure water. Add 10 μl of sodium acetate and 250 μl of 99.5% (v/v) ethanol to each sample tube and mix using micro tube mixer for 60 seconds at room temperature. Centrifuge at 12,000 g for 10 minutes at 4°C and after removal of supernatant carefully, add 400μl of 75% ethanol to each sample tube. Centrifuge at 12,000 g for 10 minutes at 4°C and discard supernatant and keep RNA pellet. Dry RNA pellet in centrifuge desiccator and re-suspend it in 30 μl ultrapure water. Quantitate total RNA using the NanoDrop 1000 Spectrophotometer and prepare 200 ng/μl of total RNA.Sample Collection - Harvest plants and move plants in liquid N2 as soon as possible (sub- 10 seconds). Transfer the frozen plants (150-300 mg) to a mortar containing liquid N2 and grind to a very fine powder using pestle. The plants can be kept frozen during grinding by the addition of liquid N2. Transfer the powdered plants (~100 mg) into a pre-cooled (in liquid N2) 2 ml eppendorf tube using pre-cooled spatula and move each sample tube in liquid N2.Growth Protocol - The hydroponic experiment was used to grow DJ123 and Nipponbare (NB) under control (0.75 mM S) and low-sulfur (0.01 mM S) conditions. The sulfur concentrations in each treatment were adjusted by replacing sulfur-containing reagents with non-sulfur-containing alternatives: K₂SO₄ with KCl, MgSO₄ with MgCl₂, ZnSO₄ with ZnCl₂, and CuSO₄ with CuCl₂, based on the Yoshida solution (Yoshida et al., 1971).Labeling - Prepare 200 ng/1.5 μl of diluted total RNA, 2 μl of final diluted Spike mixture and 1.8 μl of diluted T7 promoter primer mixture in a 0.2 ml microcentrifuge tube. Each tube now contains a total volume of 5.3 μl. Incubate reactions in a thermal cycler for 10 minutes at 65°C to denature the primer and the RNA sample. Move the reactions on ice and incubate for 5 minutes and spin each sample briefly to drive down tube contents from the tube walls and lid. Add 4.7 μl of cDNA master mixture to each sample tube and mix by pipetting up and down and incubate reactions at 40°C in a thermal cycler for 2 hours. Each tube now contains a total volume of 10 μl. Incubate reactions in a thermal cycler for 15 minutes at 70°C to inactivate the AffinityScript enzyme. Move reactions to ice and incubate for 5 minutes. Spin reactions briefly to drive down tube contents from the tube walls and lid. Add 6 μl of Transcription master mixture to each sample tube. Gently mix by pipetting and incubate samples in a thermal cycler for 2 hours at 40°C. Each tube now contains a total volume of 16 μl.Scaning - Put assembled slide holders into the scanner carousel. In the Scan Control main window, choose the slot number of the first slide for Start Slot and the slot number for the last slide for End Slot. For 8x60K microarrays, select Profile AgilentG3_GX_2Color. In the Scan Control main window, click Scan Slot m-n where m is the slot of the first slide, and n is the slot for the last slide. Open the Agilent Feature Extraction (FE) software and open the images (.tif). Save the FE Project (.fep) by selecting File > Save As and browse for desired location > Start Extracting. After the extraction is completed successfully, view the QC report for each extraction set by double-clicking the QC Report link in the Summary Report tab. Determine whether the grid has been properly placed by inspecting Spot Finding at the Four Corners of the Array.17 hours.Hybridization - Add 600 ng of cyanine 3-labeled, linearly amplified cRNA linearly amplified cRNA, 5 μl of diluted 10X Blocking Agent and 1 μl of 25X Fragmentation Buffer and gently mix by pipetting. Prepare the reactions for a total volume of 25 μl. Incubate at 60°C for exactly 30 minutes to fragment RNA. Move the reactions on ice and incubate for 60 seconds. Add 25 μl of 2X GEx Hybridization Buffer HI-RPM to stop the fragmentation reaction and mix well by careful pipetting. Take care to avoid introducing bubbles. Do not mix using vortex mixer. Centrifuge at 12,000 g for 60 seconds at room temperature to drive down tube contents from the tube walls and lid. Use immediately. Do not store. Move sample on ice and load onto the array as soon as possible. Load a clean gasket slide into the Agilent SureHyb chamber base with the label facing up and aligned with the rectangular section of the chamber base. Ensure that the gasket slide is flush with the chamber base and is not ajar. Slowly dispense 40μl of hybridization sample onto the gasket well in a “drag and dispense” manner. Slowly place an array “active side” down onto the SureHyb gasket slide, so that the “Agilent”-labeled barcode is facing down and the numeric barcode is facing up. Make sure the sandwich-pair is properly aligned. Place the SureHyb chamber cover onto the sandwiched slides and slide the clamp assembly onto both pieces. Hand-tighten the clamp onto the chamber. Vertically rotate the assembled chamber to wet the gasket and assess the mobility of the bubbles. If necessary, tap the assembly on a hard surface to move stationary bubbles. Place assembled slide chamber in rotisserie in a hybridization oven set to 65°C. Set your hybridization rotator to rotate at 10 rpm when using 2X GEx Hybridization Buffer HI-RPM. Hybridize at 65°C for 17 hours.MIAME ScoreRaw DataOrganizationAssays and DataMAGE-TAB FilesArray DesignsKyonoshin MaruyamafalseTranscription profiling of DJ123 and Nipponbare rice varieties to low-sulfate hydroponic conditionsWe conducted an oligo microarray analysis to investigate the gene transcripts in the roots of NB and DJ123 grown in LS and control hydroponic conditions2026-06-01T00:00:00Z2026-06-01T01:01:08.701Z2025-04-07T10:13:07.279ZE-MTAB-15013EFO_0002768EFO_0002944EFO_0003814EFO_0003813EFO_0003789EFO_0005518EFO_0003815