|
| Status |
Public on Jan 27, 2023 |
| Title |
Normal |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Normal pancreas
|
| Organism |
Mus musculus |
| Characteristics |
gender: male
strain: C57BL/6
age: 7 weeks
tissue: pancreas
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Pancreatic cancerous tissues from gene-modified mice and normal pancreas tissues from control mice were frozen in liquid nitrogen immediately after resection and stored at −80℃. Frozen tissues were crushed without thawing using the SK mill (Tokken, Chiba, Japan) and immediately immersed in ice-cold Isogen reagent for RNA extraction (Nippon Gene, Tokyo, Japan).
|
| Label |
Hy3
|
| Label protocol |
CIP reaction （miRCURY LNA microRNA Array Hi-Power Labeling kit） Total RNA: 250ng - 1,000 ng, Nuclease Free Water → Total 3 μL ** over 84 ng/μL ** (Mixture: 1 rxn.) ----------------------------- CIP Buffer: 0.5 μL Spike-in control: 1 μL CIP enzyme: 0.5 μL ----------------------------- Total: 2 μL Incubate at 37 ℃ for 30 min, at 95 ℃ for 5 min. on ice for 2–15 min. Labeling （miRCURY LNA microRNA Array Hi-Power Labeling kit） (Mixture: 1 rxn.) ------------------------------- Labeling Buffer: 3 μL DMSO: 2 μL Labeling enzyme: 1 μL ------------------------------- Total: 6 μL Add CIP reaction: 5 μL Fluorescent label: 1.5 μL (Sample: Hy3, Reference: Hy5) Incubate at 16 ℃ for 2 hr, at 65 ℃ 15 min.
|
| |
|
| Channel 2 |
| Source name |
reference(blends equal amounts of each samples)
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Pancreatic cancerous tissues from gene-modified mice and normal pancreas tissues from control mice were frozen in liquid nitrogen immediately after resection and stored at −80℃. Frozen tissues were crushed without thawing using the SK mill (Tokken, Chiba, Japan) and immediately immersed in ice-cold Isogen reagent for RNA extraction (Nippon Gene, Tokyo, Japan).
|
| Label |
Hy5
|
| Label protocol |
CIP reaction （miRCURY LNA microRNA Array Hi-Power Labeling kit） Total RNA: 250ng - 1,000 ng, Nuclease Free Water → Total 3 μL ** over 84 ng/μL ** (Mixture: 1 rxn.) ----------------------------- CIP Buffer: 0.5 μL Spike-in control: 1 μL CIP enzyme: 0.5 μL ----------------------------- Total: 2 μL Incubate at 37 ℃ for 30 min, at 95 ℃ for 5 min. on ice for 2–15 min. Labeling （miRCURY LNA microRNA Array Hi-Power Labeling kit） (Mixture: 1 rxn.) ------------------------------- Labeling Buffer: 3 μL DMSO: 2 μL Labeling enzyme: 1 μL ------------------------------- Total: 6 μL Add CIP reaction: 5 μL Fluorescent label: 1.5 μL (Sample: Hy3, Reference: Hy5) Incubate at 16 ℃ for 2 hr, at 65 ℃ 15 min.
|
| |
|
| |
| Hybridization protocol |
Purification Mix Hy3, Hy5 labeled RNA and add 75 μL of Nuclease Free water. Add 10 μL of 3 M NaOAc and mix well. Add 110 μL of Isopro Centrifuge at 14,000 g at 4 ℃ for 20 min. Discard sup., add 500 μL of 80 ％ EtOH, and centrifuge at 14,000 g at 4 ℃ for 5 min. Discard sup., and dry out for 3 min with speed-vac. Elute with 4 μL DMSO, 15 μL Nuclease Free water, 6 μL Labeling Buffer Add 25 μL of “2 x Hybridization Buffer” (pre-heated at 56 ℃). Hybridization Denature the solution at 95 ℃ for 5 min. Centrifuge at 12,000 g at 4 ℃ for 2 min. Add 30 μL of 20 x salt Buffer to ditch of “Hybridization Chamber”. Slide is capped by “MASTUNAMI Gap Cover Glass (CG00044 or 24*60 25PCS)”. Fill the gap with 45 μL of the solution. Incubate in 56 ℃ water bath for 16-20 hr.
|
| Scan protocol |
Wash Separate gap cover glass from the slide in “Wash buffer A” (pre-heated at 56 ℃). Wash with “Wash buffer A” (pre-heated at 56 ℃) for 2 min. Wash briefly (one plunge) with “Wash buffer B”. Wash with “Wash buffer B” for 2 min. Wash with “Wash buffer C” for 2 min. Centrifuge at 1000 rpm for 5 min. Scan Scanner; G2505C (Agilent) Software; Agilent Scan Control (Agilent) Green and Red PMT: 100 %, Scan resolution: 10 um,
|
| Data processing |
Data Analysis Software; Feature Extraction 10.7.3.1 (Agilent) ・Probe Name ・Gene Name ・rProcessed Signal (Hy5) ・gProcessed Signal (Hy3) Calculate the average of replicated spots (Hy3 or Hy5 data). Calculate the average of Hy3/Hy5 of each replicated spots (Normalized data).
|
| |
|
| Submission date |
Dec 20, 2022 |
| Last update date |
Jan 28, 2023 |
| Contact name |
Takahiro Seimiya |
| E-mail(s) |
seimiya.takahiro@gmail.com
|
| Organization name |
The university of Tokyo
|
| Street address |
7-3-1 Hongo
|
| City |
Bunkyoku |
| State/province |
Tokyo |
| ZIP/Postal code |
113-8655 |
| Country |
Japan |
| |
|
| Platform ID |
GPL17382 |
| Series (1) |
| GSE221449 |
microRNA expression in KrasG12D+Tgfbr2-/- mouse pancreatic cancer |
|
