Total RNA was extracted from tissues using miRNeasy mini kit (Qiagen AB). MicroRNA differential expression analysis was performed using the miRCURY LNA™ Array v. 18.0 (Exiqon; Qiagen, Inc.) as previously described (24 (link)). Briefly, total RNA was labeled using miRCURY™ Hy3™/Hy5™ power labeling kit (Exiqon; Qiagen, Inc.) and hybridized on miRCURY™ LNA Array (v 1.8.0). After washing and staining, the microarray slides were scanned in an Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc.). Scanned images were then imported into GenePix Pro 6.0 software (Molecular Devices, LLC) for grid alignment and data extraction. Differently expressed genes were then identified through fold-change (fold-change ≥2) and P-value (P<0.05). Subsequently, the miRNAs were measured by Volcano Plot filtering using GraphPad Prism 7.0 software package (GraphPad Software, Inc.). Finally, a hierarchical cluster heatmap representing expression intensity and direction was created using a method of hierarchical clustering via GeneSpring GX, version 7.3 (Agilent Technologies, Inc.).
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MicroRNA Expression Analysis Protocol
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Other organizations : Renji Hospital, Shanghai Jiao Tong University
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Variable analysis
independent variables
- MiRCURY LNA™ Array v. 18.0 (Exiqon; Qiagen, Inc.)
- MicroRNA differential expression
- Total RNA extraction using miRNeasy mini kit (Qiagen AB)
- Total RNA labeling using miRCURY™ Hy3™/Hy5™ power labeling kit (Exiqon; Qiagen, Inc.)
- Microarray hybridization on miRCURY™ LNA Array (v 1.8.0)
- Microarray scanning in an Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc.)
- Grid alignment and data extraction using GenePix Pro 6.0 software (Molecular Devices, LLC)
- Volcano Plot filtering using GraphPad Prism 7.0 software package (GraphPad Software, Inc.)
- Hierarchical cluster heatmap analysis using GeneSpring GX, version 7.3 (Agilent Technologies, Inc.)
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