Computational protocol: Modulation of miRNAs by Vitamin C in Human Bone Marrow Stromal Cells

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Protocol publication

[…] The microRNA array was performed only on samples treated with the high dose (100 µM) of vitamin C. miRNAs were isolated using an miRNA isolation kit (SABiosciences Corporation, Frederick, MD, USA) that specifically captures small RNAs with length of less than 200 nucleotides as per the manufacturer’s protocol. RNA concentrations were determined using a NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The quality of RNA samples was characterized on an Agilent BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) using an RNA6000 Nano Chip (Agilent). Microarrays were performed on miRNA using an Affymetrix GeneChip® miRNA 2.0 array at the Integrated Genomics Core, Augusta University, GA, USA. Details of the procedure can be found online at http://www.augusta.edu/cancer/research/shared/genomics. The miRNA profile was analyzed for hierarchical clustering of miRNA to generate heat maps. The results were normalized using robust multichip averages. T-tests were used to calculate the p-value to determine whether there is a significant difference for miRNA expression between the control and the treatment groups. Principal component analysis (PCA) was performed between vitamin C treatment and control samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analyses were performed using DIANA-miRPath v. 3.0 (http://diana.imis.athena-innovation.gr/DianaTools/index.php) on differentially expressed microRNAs target genes. GO word clouds were generated using the online Wordle software (www.wordle.net). Bioinformatics software (http://www.targetscan.org/vert_71/ and http://www.mirdb.org/) was used to predict targets genes of differentially regulated miRNAs of musculoskeletal importance.Validation of miRNA using real time-PCR: Two hundred nanograms of enriched small RNA were converted into cDNA using RT2 miRNA First Strand Kit (SABiosciences Corporation, Frederick, MD, USA). Fifty picograms of cDNA were amplified in each qRT-PCR using syber green dye and miRNA specific primers. The real-time qRT-PCR was performed on a Bio-rad q-pcr machine with following cycling parameters: 95 °C for 10 min, then 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. SYBR Green fluorescence was recorded during the annealing step of each cycle. The average of RNU6 (RNA, U6 small nuclear 2) and SNORD (small nucleolar RNA, C/D box) was used as normalization reference genes for miRNAs. Relative expression of miRNA was evaluated by using the comparative cycle threshold (CT) method (ΔΔCt). […]

Pipeline specifications

Software tools DIANA-miRPath, TargetScan
Databases miRDB KEGG
Application miRNA array analysis
Organisms Homo sapiens
Chemicals Ascorbic Acid, Nucleotides