9 protocols
ID_REF = VALUE = log2 quantile normalized Detection_Call =
Initial QC and processing was done using Affymetrix GeneChip Operating Software(GCOS) to obtain the raw signal intensities. The data were analyzed with Avadis using the default analysis settings and quantile normalization as normalization method afer being log transformed.
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
Following fragmentation, 20 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Zebrafish Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
1-2 cell stage embryos were collected for microinjection. Following injections the embryos were aged at 30C and were lated collected at the different time points (1hpf, 7hpf and 24hpf)
Total RNA was extracted using Trizol as per the manufacturer's instructions.
Embryos were collected at one cell stage. Embryos were injected with anti-mir-34LNA of mockLNA at the 1-2 cell stage and were harvetyes at the specified time points of 1hpf, 7hpf and 24hpf. The embryos were also monitoredunder the dissecting microscope for any developmental abnormalities of phenotypic defects. Selected embryos were transferred to a fresh petriplate. The injected embryos at 7hpf and 24hpf were dechorionated manually, rinsed with distilled water and placed on ice in the Trizol solution (GibcoBRL).