All cutaneous specimens were harvested in the operating room, immediately stored in RNAlater (Quiagen, Hilden, Germany) and kept at - 80 °C until RNA isolation.
Extracted molecule
total RNA
Extraction protocol
Total RNA including miRNAs was isolated using the miRNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturer’s protocol. RNA concentration and purity was determined by NanoDrop ND-1000 spectral photometer (Peqlab, Erlangen, Germany). RNA integrity control (RIN) was determined by means of capillary electrophoresis with the 2100 Bioanalyzer and the RNA 6000 NanoLabCHip Kits (both Agilent Technologies, Santa Clara, USA).
Label
Cy3
Label protocol
For assessment of labeling and hybridization efficiencies total RNA samples were spiked with in-vitro synthesized oligonucleotides by using the MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA). A total of 100 ng total RNA per sample were introduced into a labeling reaction. The spiked total RNA was treated with alkaline calf intestine phosphatase (CIP). The dephosphorylated RNA was labeled with the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, USA) according to the manufacturer´s instructions using T4 RNA ligase, incorporating Cyanine 3-Cytidine biphosphate (pCp). The Cyanine-3-labeled miRNA samples were prepared for One-Color based hybridization with Complete miRNA Labeling and Hyb Kit (Agilent Technologies, Santa Clara, USA) according to the manufacturer´s instructions.
Hybridization protocol
Labeled miRNA samples were hybridized at 55°C for 20 hrs on separate Human miRNA Microarrays Release 16.0 (8x60K format, (Agilent Technologies, Santa Clara, USA). Afterwards, microarrays were washed with increasing stringency using Gene Expression Wash Buffers (Agilent Technologies, Santa Clara, USA) followed by drying with acetonitrile (Sigma-Aldich, St.Louis, USA).
Scan protocol
Fluorescent signal intensities were detected with Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA) on the Agilent DNA Microarray Scanner.
Description
RS-227_06.txt
Data processing
Fluorescent signal intensities were extracted from the images using Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA). All the steps described were carried out according to the manufacturer´s instructions.
Submission date
Dec 14, 2011
Last update date
Nov 02, 2012
Contact name
Michael Sand
Organization name
Ruhr-University Bochum
Department
Depatment of Dermatology, Venereology and Allergology
