Small rna nano labchip

Total RNA was isolated from RMS cell lines using Trizol Reagent (Life Technologies) following the manufacturer’s protocol. The commercially available PureLink miRNA Isolation Kit (Life Technologies) was used to enrich total RNA preparation for small RNA molecules (< 200 bp). Total and small RNA were quantified using the ND-1000 spectrophotometer (Nanodrop, Wilmington, DE); RNA integrity and the content of microRNAs (%) in each sample were assessed by capillary electrophoresis utilizing the RNA 6000 Nano LabChip and the Small RNA Nano LabChip, respectively, using the Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Only total RNA samples with RNA Integrity Number (R.I.N.) values higher than 7 and the percentage of miRNA < 20% were used for microarray analysis.

Total RNA, including miRNAs, was extracted from olfactory epithelium cells by using Monarch^® Total RNA Miniprep kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Total RNA was quantified by NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity and content of miRNAs (%) in each sample were assessed by capillary electrophoresis with the RNA 6000 Nano LabChip and the Small RNA Nano LabChip, respectively, by using the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Both nostrils were sampled independently, and the obtained cells were used for total RNA extraction (Supplementary Figure 1). For each subject, only the best sample in terms of RNA quantity and quality was selected and used for further analysis. More detailed information about RNA samples is reported in Table 1. Only samples with RNA Integrity Number (R.I.N.) values higher than six and a percentage of miRNA, calculated on the total of small RNA, between 40% and 65% were used for gene and miRNA expression analysis.

For the microarray experiments mRNA and miRNAs were isolated from frozen gastrocnemius muscles. miRNAs/mRNAs isolation was achieved by an initial purification with TRIzol (Invitrogen) followed by subsequent purification and fractionation with the Purelink miRNA Isolation Kit (Invitrogen number K1570-01). Total and small RNA were quantified using the ND-1000 spectrophotometer (Nanodrop), whereas RNA integrity and content of microRNAs (%) in each sample were assessed by capillary electrophoresis using the RNA 6000 Nano LabChip and the Small RNA Nano LabChip, respectively, using the Agilent Bioanalyzer 2100 (Agilent Technologies). Only total RNA samples with RNA integrity number values >6, and the percentage of miRNA <20% were used for microarray analysis.