|
| Status |
Public on Dec 01, 2014 |
| Title |
N2 on control RNAi replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
whole organism total RNA isolated, Control RNAi treated, replicate-1
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
larvae stage: L4 stage
|
| Treatment protocol |
Strains for RNA isolation were initially grown on E. coli OP50. Gravid adult worms were bleached and the eggs were hatched on plates containing respective RNAi bacteria. For sample collection L4 staged worms were washed off the plates with M9 buffer.
|
| Growth protocol |
RNAi plates were prepared by supplementing Nematode Growth Media (NGM) media with 100 µg/ml ampicillin and 2 mM IPTG. RNAi bacteria were grown overnight at 37°C in LB media supplemented with 100 µg/ml ampicillin and 12.5 µg/ml tetracycline. The next day, the cultures were diluted (1:50) in LB containing 100 µg/ml ampicillin and grown at 37°C until an OD600 of 0.9. The bacterial pellets were resuspended in 1X PBS (phosphate-buffered saline) containing 1 mM IPTG. About 200 µl of the bacterial suspension was seeded onto the RNAi plates. The seeded plates were dried at RT for 2-3 days and stored at 4°C till further use.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Trizol (Invitrogen, USA). Briefly, worms grown on vector or RNAi of interest were washed off the plates with M9 buffer. Then, 0.3 ml of Trizol reagent was added and the worms lysed by vigorous vortexing. RNA was purified by phenol:chloroform:isoamylalcohol extraction and ethanol precipitation. The concentration and the purity of the RNA were determined using Bioanalyzer (Agilent, USA). Alternatively, the quality of the ribosomal 28 S and 18 S on an agarose gel was used as a measure of integrity and the absorbance at 260/280 nm was used to determine quantity.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
Standard Agilent procedures
|
| Scan protocol |
Standard Agilent procedures
|
| Description |
Gene expression of control RNAi treated N2 worms at L4 stage
|
| Data processing |
Feature Extraction Software 9.1 (Agilent)
|
| |
|
| Submission date |
Aug 21, 2012 |
| Last update date |
Dec 01, 2014 |
| Contact name |
Arnab Mukhopadhyay |
| E-mail(s) |
arnab@nii.ac.in
|
| Phone |
+91 11 26703885
|
| Organization name |
National Institute of Immunology
|
| Lab |
Molecular Aging Lab
|
| Street address |
Aruna Asaf Ali Marg
|
| City |
New Delhi |
| ZIP/Postal code |
110067 |
| Country |
India |
| |
|
| Platform ID |
GPL11346 |
| Series (1) |
| GSE40252 |
Gene expression profile of C. elegans worms when mekk-3 is knocked down by RNAi |
|
