Punch biopsies (4 mm) were obtained under local anaesthesia and immediately put in RNAlater (Qiagen, Hilden, Germany) and stored at - 80 °C until RNA extraction.
Growth protocol
A total of 6 patients with BCC and 6 healthy individuals (control; non-lesional skin) were enrolled in the study.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with TRIzol (Life Technologies, Carlsbad, USA) following the manufacturers protocol. RNA quantity and quality was determined with NanoDrop ND-1000 and RNA integrity with standard denaturing agarose gel electrophoresis. To purify mRNA from total RNA, rRNA was removed with mRNA-ONLY™ Eukaryotic mRNA Isolation Kit (Epicentre, Omaha, USA).
Label
CY3
Label protocol
The samples were amplified with a mixture of oligo (dT) and random priming method (Arraystar Flash RNA Labeling Kit, Arraystar) and transcribed in fluorescent cRNA which was purified by RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers protocol. Concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) was measured with NanoDrop ND-1000. Next, to 1 μg of each labeled cRNA, 5 μl 10 × Blocking Agent (Agilent Technology, Santa Clara, USA) and 1 μl of 25 × Fragmentation Buffer was added (Agilent Technology, Santa Clara, USA). The mixture was heated at 60 °C for 30 min and 25 μl 2 × GE Hybridization buffer (Agilent Technology, Santa Clara, USA) was added to dilute the labeled cRNA.
Hybridization protocol
For hybridization 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarrays which were incubated in an Agilent hybridization oven (Agilent Technology, Santa Clara, USA) for 17 hours at 65°C.
Scan protocol
Finally, arrays were washed, fixed and scanned with Agilent DNA Microarray Scanner G2505C (Agilent Technologies, Santa Clara, USA).
Description
A total of 1851 LncRNAs were shown to be significantly up- and 2165 LncRNAs to be significantly down-regulated with the criteria of FC ≥2 and p<0.05) in BCC compared to mon-lesional skin (control).
Data processing
Quantile normalization which was applied to raw data and further data processing was performed with GeneSpring GX v12.0 software package (Agilent Technologies, Santa Clara, USA). Only lncRNAs and mRNAs that at least in 3 out of 6 samples have flags in Marginal or Present (“All Targets Value”) were chosen for further data analysis. Hierarchical clustering was performed with R software (version 2.15, http://www.r-project.org/) in order to arrange samples into groups based on their expression levels.
Submission date
Nov 10, 2015
Last update date
Mar 29, 2016
Contact name
Michael Sand
Organization name
Ruhr-University Bochum
Department
Depatment of Dermatology, Venereology and Allergology
