|
| Status |
Public on Dec 05, 2012 |
| Title |
PSG at M4, biological rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
silkworm PSG at M4
|
| Organism |
Bombyx mori |
| Characteristics |
strain: p50
tissue: posterior silk gland (PSG)
Stage: M4
|
| Treatment protocol |
The dissected PSGs were rinsed thrice in ice-cold RNase-free physiological saline, and then immediately froze with liquid nitrogen and stored at -80 °C.
|
| Growth protocol |
Silkworm strain p50 was reared on the fresh mulberry leaves under a 12 h light/12 h dark photoperiod at 26 ± 1 °C with 70–85% relative humidity.The developmental stages were synchronized after fourth molt by collecting new larvae.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Two micrograms of total RNA were used to prepare the fluorescent dye-labeled cDNA using the linear mRNA amplification procedure and a further Klenow enzyme labeling method. Dual-dye experiments were performed to analyze the gene expression patterns of PSG at M4, V1, V3, V5, and W; a total of 15 samples were tested including three biological replicates at each time point. An aliquot of the test samples were pooled as a common reference sample (CKA). The test samples and CKA were labeled with Cy3, Cy5, respectively. Cy5-dCTP, Cy3-dCTP (GE Healthcare Cat. No. PA 55021/ PA 53021).
|
| |
|
| Channel 2 |
| Source name |
common refference (CKA)
|
| Organism |
Bombyx mori |
| Characteristics |
strain: p50
Stage: Aliquots of each test sample were pooled as a common reference sample (CKA)
tissue: posterior silk gland (PSG)
|
| Treatment protocol |
The dissected PSGs were rinsed thrice in ice-cold RNase-free physiological saline, and then immediately froze with liquid nitrogen and stored at -80 °C.
|
| Growth protocol |
Silkworm strain p50 was reared on the fresh mulberry leaves under a 12 h light/12 h dark photoperiod at 26 ± 1 °C with 70–85% relative humidity.The developmental stages were synchronized after fourth molt by collecting new larvae.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
Two micrograms of total RNA were used to prepare the fluorescent dye-labeled cDNA using the linear mRNA amplification procedure and a further Klenow enzyme labeling method. Dual-dye experiments were performed to analyze the gene expression patterns of PSG at M4, V1, V3, V5, and W; a total of 15 samples were tested including three biological replicates at each time point. An aliquot of the test samples were pooled as a common reference sample (CKA). The test samples and CKA were labeled with Cy3, Cy5, respectively. Cy5-dCTP, Cy3-dCTP (GE Healthcare Cat. No. PA 55021/ PA 53021).
|
| |
|
| |
| Hybridization protocol |
The labeled cDNA were hybridized with the hybridization solution (3×SSC, 0.2%SDS, 5×Denhart’s, 25% methanamide) for 16 hr at 42 °C on the 23K Silkworm Genome Arrays (CapitalBio Corp., Beijing, China). The arrays were washed with 0.2% SDS，2×SSC at 42 °C for 5 min followed by another 5 min washing with 0.2×SSC at room temperature.
|
| Scan protocol |
Array signals were scanned with a LuxScanTM 10K/A dual channels laser scanner and the images obtained were then analyzed using LuxScan™ 3.0 software (both from CapitalBio Corp.).
|
| Description |
The test samples and CKA were labeled with Cy3 and Cy5, respectively.
|
| Data processing |
The data were analyzed with LuxScanTM 3.0 software (CapitalBio Corp.). The extracted signal data was filtered by removing faint spots, saturated spots (intensity >60,000), and signal intensities below 800 units after subtracting the background from both channels (Cy3 and Cy5). The prevalent LOWESS normalization method was applied for the checked signal intensity data.
After discarding the five housekeeping genes (including sw22934, sw13956, sw15061, sw01783, sw08699), exogenous controls (including Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8), immobilized control (Hex), and negative control (50%DMSO), the extracted signal data was filtered by removing faint spots, saturated spots (intensity >60,000), and signal intensities below 800 units after subtracting the background from both channels (Cy3 and Cy5). The prevalent LOWESS normalization method was applied for the checked signal intensity data. The active transcript is defined as that the signal intensities of at least two biological replicates at one stage have to pass the thresholds.
|
| |
|
| Submission date |
Dec 19, 2010 |
| Last update date |
Dec 05, 2012 |
| Contact name |
Bo-xiong Zhong |
| E-mail(s) |
bxzhong@zju.edu.cn
|
| Organization name |
College of Animal Sciences, Zhejiang University
|
| Street address |
268 Kaixuan Road
|
| City |
Hangzhou |
| ZIP/Postal code |
310029 |
| Country |
China |
| |
|
| Platform ID |
GPL10763 |
| Series (1) |
| GSE26172 |
Expression data from silkworm posterior silk glands at the late larval stages |
|
