|
| Status |
Public on Aug 25, 2017 |
| Title |
UF_Midgut_4 |
| Sample type |
RNA |
| |
|
| Source name |
mosquito female midgut; sugarfed; replicate 8
|
| Organism |
Anopheles gambiae |
| Characteristics |
Sex: female
tissue: Midgut
condition: sugarfed
|
| Treatment protocol |
Three-day-old virgin A. gambiae females were fed with either uninfected human blood, P. falciparum-infected blood or 10% sucrose solution. All mosquito groups were kept at 26°C and 80% humidity. At 18 h post feeding, female mosquitoes were dissected on ice to collect head, midgut, ovaries and the abdominal carcass. The latter includes the fat body as well as the cuticle. Per time point and sample, tissues of 15 female mosquitoes were pooled in cold PBS. After sample collection, the PBS was removed and 500 µl TRIzol added to the tissue samples. The tissues were homogenized using steal beads in a Qiagen tissue lyser (8 min of 50 rpm at 4°C) and kept at -80°C until further usage. The experiment was performed in four biological replicates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s recommendations. Briefly, after phase separation by centrifugation total RNA was purified by silica membrane, eluted with water and stored at -80°C. The total RNA yield was measured by NanoDropND-1000 Spectrophotometer. The integrity of total RNA was assessed with a 2100 Bioanalyzer and a RNA 6000 Nano LabChip kit. Furthermore, the ratio of miRNA fraction to small RNAs from isolated total RNA was monitored by use of the Agilent Small RNA kit.
|
| Label |
Cy3
|
| Label protocol |
An amount of 100 ng total RNA was processed with the miRNA Complete Labeling and Hyb Kit according the supplier’s recommendations. In brief, samples were dephosphorylated with calf intestine alkaline phosphatase and further denatured with DMSO. The sample RNA functioned as acceptor in a T4 RNA ligase mediated reaction with the RNA donor 3’, 5’-cytidine bisphosphate having a single Cy3 label attached to the 3’ phosphate of the nucleotide (Cy3-pCp).
|
| |
|
| Hybridization protocol |
Labeling reactions were column purified, dried down using a speed-vac at 45°C and resuspended in blocking and hybridization buffer.
|
| Scan protocol |
After scanning at 5 µm resolution with a G2565CA high-resolution laser microarray scanner features were extracted with an image analysis tool version A.11.5.1.1 using the default protocol
|
| Data processing |
Microarray data were analysed using the R limma package. All arrays were corrected for background using the normexp function and normalized by quantile normalization. Next, the data of all replicated spots on the array were pooled using the avereps function, which resulted in an elist containing log2 relative expression values
|
| |
|
| Submission date |
Aug 24, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Lena Lampe |
| Organization name |
Max-Planck-Institute for Infection Biology
|
| Street address |
Charitéplatz 1
|
| City |
Berlin |
| ZIP/Postal code |
13357 |
| Country |
Germany |
| |
|
| Platform ID |
GPL23950 |
| Series (1) |
| GSE103034 |
miRNA expression in Anopheles gambiae tissues |
|
