|
| Status |
Public on May 21, 2020 |
| Title |
Liver cancer 3 |
| Sample type |
RNA |
| |
|
| Source name |
Liver cancer
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Liver cancer
age: one-year-old
|
| Extracted molecule |
total RNA |
| Extraction protocol |
using the Biopulverizer(biospec) and the Mini-Bead-Beater-16(biospec) to make tissues into the homogenized samples, and then incubate the homogenized samples for 5 minutes at 15 to 30°C to permit the complete dissociation of nucleoprotein complexes. Add 0.2 ml of chloroform per 1 ml of TRIzol® Reagent (Invitrogen life technologies). Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at 15 to 30°C for 2 to 3 minutes. Centrifuge the samples at 12,000 × g for 15 minutes at 4°C. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIzol Reagent used for homogenization.
|
| Label |
Cyanine-3-CTP
|
| Label protocol |
Using Quick Amp Labeling Kit, One-Color (Agilent p/n 5190-0442).Add 1ug of total RNA to a 1.5-mL microcentrifuge tube.Add 1.2 μL of T7 Promoter Primer.Use nuclease-free water to bring the total reaction volume to 11.5 μL.Denature the primer and the template by incubating the reaction at 65°C in a circulating water bath for 10 minutes.Place the reactions on ice and incubate for 5 minutes.Immediately prior to use, gently mix the components including 5×First Strand Buffer,0.1M DTT and 10mM dNTP mix etc. in the following table for the cDNA Master Mix by adding in the order indicated, and put on ice.Briefly spin each sample tube in a microcentrifuge to drive down the contents from the tube walls and the lid. Return the tubes to ice. Add 8.5 μL of cDNA Master Mix to each sample tube and mix by pipetting up and down.Incubate samples at 40°C in a circulating water bath for 2 hours.Move samples to a 65°C circulating water bath and incubate for 15 minutes.Move samples to ice. Incubate for 5 minutes.Spin samples briefly in a microcentrifuge to drive down tube contents from the tube walls and lid.Immediately prior to use, gently mix the components including T7 RNA Polymerase and Cyanine-3-CTP etc. in the order indicated for the Transcription Master Mix by pipetting at room temperature.Add 60 μL of Transcription Master Mix to each sample tube. Gently mix by pipetting.Incubate samples in a circulating water bath at 40°C for 2 hours.
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| |
|
| Hybridization protocol |
1. Add 500 μL of nuclease-free water to the vial containing lyophilized 10X Blocking Agent. Mix by gently vortexing. 2. Equilibrate water bath to 60°C. 3. For 8*60K array, add each of the components as indicated in the tables as below to a 1.5 mL nuclease-free microfuge tube:cyanine 3-labeled, linearly amplified cRNA 0.6ug,10X Blocking Agent 5ul,Nuclease-free water ×ul,25X Fragmentation Buffer 1ul.4.Mix well but gently on a vortex mixer. 5. Incubate at 60°C for exactly 30 minutes to fragment RNA. 6. Add 2x GEx Hybridization Buffer HI-RPM to the array to stop the fragmentation reaction.7.Mix well by careful pipetting. Take care to avoid introducing bubbles. Do not mix on a vortex mixer; mixing on a vortex mixer introduces bubbles. 8. Spin for 1 minute at room temperature at 13,000 rpm in a microcentrifuge to drive the sample off the walls and lid and to aid in bubble reduction. 9. Place sample on ice and load onto the array as soon as possible.10. 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.11. Slowly dispense the volume of hybridization sample onto the gasket well in a “drag and dispense”manner.12.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. Verify that thesandwich-pair is properly aligned.13. Place the SureHyb chamber cover onto the sandwiched slides and slide the clamp assembly onto both pieces.14. Hand-tighten the clamp onto the chamber.15. Vertically rotate the assembled chamber to wet the gasket and assess the mobility of the bubbles. 16. Place assembled slide chamber in rotisserie in a hybridization oven set to 65°C. Set your hybridization rotator to rotate at 10 rpm. 17. Hybridize at 65°C for 17 hours.
|
| Scan protocol |
1. Assemble the slides into an slide holder. 2. Place assembled slide holders into scanner carousel. 3. Verify scan settings for one-color scans. Scan region:Scan Area (61 x 21.6 mm);Scan resolution (μm): 5;5μm scanning mode: Single Pass;eXtended Dynamic range:(selected);Dye channel:Green;Green PMT: XDR Hi 100% ,XDR Lo 10%. 4. Click Scan Slot m-n on the Scan Control main window where the letter m represents the Start slot where the first slide is located and the letter n represents the End slot where the last slide is located.
|
| Data processing |
Extract data using Agilent Feature Extraction Software. Software:Agilent Feature Extraction. Procedure: 1. Open the Agilent Feature Extraction (FE) software.2. Add the images (.tif) to be extracted to the FE Project.3. Set FE Project Properties.4. Check the Extraction Set Configuration.5. Save the FE Project (.fep) by selecting File > Save As and browse for desired location.6. Select Project > Start Extracting and export data to txt.
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| |
|
| Submission date |
May 22, 2017 |
| Last update date |
May 21, 2020 |
| Contact name |
Yan Liu |
| E-mail(s) |
15026971620@163.com
|
| Phone |
15026971620
|
| Organization name |
Second Military Medical University, Shanghai 200433, China
|
| Department |
The Department of Medical Genetics
|
| Lab |
The Department of Medical Genetics
|
| Street address |
800 Xiangyin Road, Shanghai 200433
|
| City |
shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
200433 |
| Country |
China |
| |
|
| Platform ID |
GPL19286 |
| Series (1) |
| GSE99157 |
the lncRNA expression profiles in fetal liver, adult liver and liver cancer tissues from mice |
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