Total RNA was extracted from placental tissue using the miRVana Isolation Kit (Ambion). Protocol was used in such a way that small RNAs were retained.
Label
Biotin
Label protocol
Samples were processed by Asuragen Services (Austin,TX), according to the company’s standard operating procedures. Briefly, samples were biotinylated and purified using BioSpin6 (Bio-Rad, Hercules CA).
Hybridization protocol
Hybridization, washing, staining, imaging, and signal extraction were performed according to Affymetrix-recommended procedures, except that the 20X GeneChip Eukaryotic Hybridization Control cocktail was omitted from the hybridization.
Scan protocol
Scanned as per Asuragen's standard operating procedures.
Description
A0008645.CEL
Data processing
Two overlapping probes were designed for each mature miRNA candidate. The staggered probe design provides the flexibility required for probes to capture slight variations in the ends of the processed mature miRNA. For computationally predicted miRNA, this probe design strategy accounts for the uncertainty associated with predicting the precise cleavage site of the processed mature miRNA. The signal processing implemented for the Ambion miRChip is a multi-step process involving probe specific signal detection calls, background estimate and correction, constant variance stabilization and either array scaling or global normalization. For each probe, an estimated background value is subtracted that is derived from the median signal of a set of G-C-matched anti-genomic controls. Arrays within a specific analysis experiment were normalized together according to the variance stabilization method described by Huber et al. (Huber et al., 2002). Based on historical performance of the probes on a training data set, each of the two probes is assigned to either A or B bin. Variance Stabilization Normalization (VSN) technique is used to normalize the data. A probes are used for data interpretation.
