RNA was extracted from whole blood using the PAXgene™ Blood RNA System Kit following manufacturer's guidelines. Briefly, the samples were removed from -80°C and incubated at room temperature for 2 hours to ensure complete lysis. After that the tubes were centrifuged for 10 min at 5000 g, the supernatant decanted and 500 μL of RNase-free water added to the pellet. After washing with RNase-free water, the pellet was dissolved in 350 μl resuspension buffer and incubated with 300 μl binding buffer and 40 μl proteinase K for 10 min at 55°C in a shaker-incubator. The lysate was transferred into a PAXgene shredder spin column and centrifuged (18000 g for 3 min). The flow-through fraction was mixed with 350 μl ethanol and transferred to a PAXgene RNA spin column. After washing the column with washing buffer 1, samples were incubated with 10 μl of DNase I for 15 min. The columns were washed with washing buffer and RNA eluted with 40 μl elution buffer. The RNA yield was estimated by measuring absorbance at 260 nm in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). RNA purity was calculated from the ratio of absorbance at 260 nm and 280 nm, and RNA integrity was assessed by standard denaturing agarose gel electrophoresis.
Label
Cy3
Label protocol
Mouse LncRNA Microarray V3.0, designed by ArrayStar Inc, was employed to perform global profiling of mouse protein-coding (~24,881) transcripts. Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing a random priming method. The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000.
Hybridization protocol
1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60 °C for 30 min, finally 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled on to the microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.
Scan protocol
The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C). Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images.
Description
Control_3
Data processing
Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v11.5.1 software package (Agilent Technologies). For both mRNA and lncRNA - All Targets Value (Entities where at least 5 out of 10 samples have flags in Present or Marginal)Hierarchical Clustering was performed using the Agilent GeneSpring GX software (version 12.1).
