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| Status |
Public on Jan 29, 2015 |
| Title |
SDT-2 |
| Sample type |
RNA |
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| Source name |
melanoma_ultrasound_plus_ALA
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| Organism |
Mus musculus |
| Characteristics |
strain: BALB/c athymic nude
tissue: B16-F10 transplanted melanoma
age: 6-8 weeks
Sex: Female
status: Sacrificed
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| Treatment protocol |
When the tumor grew to a volume of ~100 mm3, approximately 5 days after implantation, mice were randomized into four groups with 12 mice per group. The groups were as follows: C, the untreated control mice; ALA, mice received 200 mg/kg ALA by intraperitoneal injection alone; US, mice treated with ultrasound (1.1 MHz, 1 W/cm2, 10 % duty cycle) alone; SDT, mice were treated with ultrasound plus ALA. After 4 hours of administration of ALA, the mice in the US and SDT groups were sonicated by ultrasound for 10 minutes in the dark. All mice were sacrificed when tumor diameter exceeded 10 millimeters after 11 days.To study the differential miRNAs expression in tissue samples, we performed miRNAs expression profiling of the tissue samples from 4 groups (Control, ALA, US, and SDT group) with 2 samples per group.
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| Growth protocol |
Female BALB/c athymic nude mice 6-8 weeks of age were obtained from SLAC Laboratory Animal Company (Shanghai, China). To create the tumor model, B16-F10 tumor cells (1 × 10^5 cells in 100 μL of serum-free culture medium) were implanted subcutaneously on to the right-side flanks of the mice.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol (Invitrogen) and miRNeasy mini kit (QIAGEN) according to manufacturer’s instructions, which efficiently recovered all RNA species, including miRNAs. RNA quality and quantity was measured by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies) and RNA Integrity was determined by gel electrophoresis.
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| Label |
Hy3
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| Label protocol |
After RNA isolation from the samples, the miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer’s guideline for miRNA labelling. One microgram of each sample was 3'-end-labeled with Hy3TM fluorescent label, using T4 RNA ligase by the following procedure: RNA in 2.0 μL of water was combined with 1.0 μL of CIP buffer and CIP (Exiqon). The mixture was incubated for 30 min at 37°C, and was terminated by incubation for 5 min at 95°C. Then 3.0 μL of labeling buffer, 1.5 μL of fluorescent label (Hy3TM), 2.0 μL of DMSO, 2.0 μL of labeling enzyme were added into the mixture. The labeling reaction was incubated for 1 h at 16°C, and terminated by incubation for 15 min at 65°C.
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| Hybridization protocol |
After stopping the labeling procedure, the Hy3TM-labeled samples were hybridized on the miRCURYTM LNA Array (v.18.0) (Exiqon) according to array manual. The total 25 μL mixture from Hy3TM-labeled samples with 25 μL hybridization buffer were first denatured for 2 min at 95°C, incubated on ice for 2 min and then hybridized to the microarray for 16–20 h at 56°C in a 12-Bay Hybridization Systems (Hybridization System - Nimblegen Systems, Inc., Madison, WI, USA), which provides an active mixing action and constant incubation temperature to improve hybridization uniformity and enhance signal. Following hybridization, the slides were achieved, washed several times using Wash buffer kit (Exiqon), and finally dried by centrifugation for 5 min at 400 rpm. Then the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA).
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| Scan protocol |
Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs that intensities>=30 in all samples were chosen for calculating normalization factor. Expressed data were normalized using the Median normalization. After normalization, significant differentially expressed miRNAs were identified through Volcano Plot filtering. Hierarchical clustering was performed using MEV software (v4.6, TIGR).
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| Description |
miRNA
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| Data processing |
The box plots are convenient way to quickly visualize the distribution of a dataset. They are most useful for comparing the distributions of samples. After normalization, the distributions of log2-ratios across every sample are nearly the same. (Left: non-normalized log2-ratio data; right: Median normalized log2-ratio data) .A correlation matrix describes correlation among replicate experiments. The scatter-plot is a visualization that is useful for assessing the variation (or reproducibility) between chips. The axes of the scatter-plot are the normalized signal values of the samples (Ratio scale).
To identify differentially expressed miRNAs with statistical significance, we performed a Volcano Plot filtering between the two groups from the experiment. The threshold we used to screen Up or Down regulated miRNAs is Fold Change>=2.0 and P-value<=0.05.
Non-normalized/Raw and normalized data are located on the series record.
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| Submission date |
Jan 28, 2015 |
| Last update date |
Jan 29, 2015 |
| Contact name |
Zheng Hu |
| E-mail(s) |
huzheng@hit.edu.cn
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| Phone |
+8613796057815
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| Organization name |
Harbin Institute of Technology
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| Street address |
92 West Dazhi Street , Nan Gang District, Harbin
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| City |
Harbin |
| ZIP/Postal code |
150080 |
| Country |
China |
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| Platform ID |
GPL17107 |
| Series (1) |
| GSE65390 |
miRNA expression-based, individualized outcome prediction for SDT treatment in melanoma |
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| Supplementary data files not provided |
| Processed data are available on Series record |
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