|
| Status |
Public on Mar 20, 2012 |
| Title |
t1 |
| Sample type |
RNA |
| |
|
| Source name |
Myeloid-derived suppressor cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: BALB/c
gender: female
age: 6-8 wk old
tumor stage: tumor-bearing mice
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was harvested using TRIzol (Invitrogen) and the RNeasy mini kit (QIAGEN).
|
| Label |
Hy3
|
| Label protocol |
Step 1: Thaw all kit components. Place all kit components on ice and thaw for 15-20 min. Mix thoroughly by vortexing followed by brief centrifugation. Do not thaw or vortex the enzymes. Flick these tubes followed by brief centrifugation. Step 2: Total RNA 2 ul; Spike-in miRNA kit v2 1ul; CIP buffer 0.5 ul; CIP enzyme 0.5 ul. Mix reagents on ice. Reagents should be combined in an RNase-free microcentrifuge tube and should be mixed by pipetting up and down to ensure that all reagents are mixed thoroughly. Step 3: Incubate 30 min. at 37°C, using a PCR cycler with heated lid. Step 4: Stop the enzyme reaction and denaturate the RNA by incubation at 95°C followed by snap cooling on ice. Step 5: Incubate 2 min on ice. Step 6: Mix reagents on ice. CIP reaction from step 5 4 ul; Labeling buffer 3 ul; Fluorescent label (Hy3™ or Hy5™) 1.5 ul; DMSO 2 ul; Labeling enzyme 2 ul. Step 7: Mix and centrifuge the reagents briefly. Step 8: Incubate for 1 hour at 16°C, using a PCR cycler with heated lid. Protect the reaction from light. Step 9: After stopping the labeling procedure, briefly spin the reaction and leave it at 4°C. The labeled sample is now ready for hybridization on the array. Hybridization should preferably occur within 1-2 h.
|
| |
|
| Hybridization protocol |
Step 1: Combine the labeled sample(s). Step 2: Add 25 μL Hybridization buffer. Step 3: Denature at 95°C for 2 min. Step 4: Incubate 2 min. on ice. Step 5: Preparation of Tecan. Step 6: Flush hyb chamber with 1x Hybridization buffer. Step 7: Inject reaction mixture. Step 8: Incubate at 56°C for 16 h. Step 9: Two runs of wash at 56°C for 1 min. using Wash buffer A. Step 10: Two runs of wash at 23°C for 1 min. using Wash buffer B. Step 11: Two runs of wash at 23°C for 1 min. using Wash buffer C. Step 12: Wash at 23°C for 30 sec using Wash buffer C. Step 13: Dry slides.
|
| Scan protocol |
We are using an Agilent G2505B Microarray Scanner System. The scanning is normally performed with 10 μm. The sensitivity should be adjusted to 100% PMT. To avoid ozone bleaching, we scan the microarrays in an ozone-free environment (less than 2 ppb ozone). Before starting any analysis, confirm that the tiff image is in the correct orientation (two landing lights in lower right corner). Depending on the scanner, the image may need to be flipped from upper left to lower right.
|
| Data processing |
The intensity of green signal is calculated after background subtraction and replicated spots on the same slide have been averaged by getting a median intensity. We use Median Normalization Method to obtain “Normalized Data”, Normalized Data=(Foreground-Background)/median, the median is 50 percent quantile of microRNA intensity which is larger than 50 in all samples after background correction.
|
| |
|
| Submission date |
Mar 06, 2012 |
| Last update date |
Mar 20, 2012 |
| Contact name |
Yang Liu |
| E-mail(s) |
immuinst@zju.edu.cn
|
| Phone |
86-571-88208281
|
| Organization name |
Zhejiang University School of Medicine
|
| Department |
Institute of Immunology
|
| Lab |
Wang QQ
|
| Street address |
388 Yu Hang Tang Road
|
| City |
Hangzhou |
| State/province |
Zhejiang |
| ZIP/Postal code |
310058 |
| Country |
China |
| |
|
| Platform ID |
GPL7723 |
| Series (1) |
|
