5 protocols
array scanning protocol
This step was done according to manufacturer's (Applied Biosystems) instructions.
(Parameters: Scanning hardware = OTHER: AbiPrism 7900 HT, Scanning software = Scanning software)
nucleic acid hybridization to array protocol
Hybridization was done by Applied Biosystems.
(Parameters: Chamber type = OTHER: TaqMan Human miRNA cards A and B, Quantity of label target used = 0, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 0)
nucleic acid extraction protocol
RNA isolation was performed with use of Exiqon RNA isolation kit. Isolation was performed according to the manufacturer’s protocol from 6-well plates (9.5 cm2 of growth area) and the amount of starting material was 1x106 cells per well. Samples were frozen at -70°C for further use in microarray experiment.
(Parameters: Extracted product = total_RNA, Amplification = PCR)
nucleic acid labeling protocol
Labeling was done by Applied Biosystems.
(Parameters: Amplification = PCR, Mass unit = Micro gram)
treatment protocol
For the wounding assay cells were seeded on 6-well plates at the initial density of 3x105 cells and cultured until confluent. Forty eight hours after reaching full confluence cells were damaged by scraping off the monolayer with a hatch-cross wounding pattern using a P200 Gilson pipette tip. After that, the medium and cell debris were removed and 2 ml of fresh serum-containing medium was added to the remaining cells. For all experiments, at least two points of reference per well of a 6-well plate were used for post-injury analyses. For miRNA expression profile 7 time points were selected.