• Mice (e.g. data presented here involves wild-type C57Bl/10 and dystrophic C57BL/10ScSn-Dmdmdx/J [mdx]) CAUTION: Experiments involving live rodents must conform to all relevant institutional and governmental regulations for animal handling and care. All animal procedures described here are permitted under PPL 30/2907 awarded to Professor Matthew J. A. Wood at the University of Oxford by the UK Home Office in accordance with UK law (Animals [Scientific Procedures] Act 1986).
• TRIzol LS Reagent (catalogue #: 10296-028 [Life Technologies]).CAUTION: Always work with TRIzol LS reagent in a fume hood. Always wear a lab coat, gloves and safety glasses.
• Chloroform (molecular biology grade)CAUTION: Always work with Chloroform in a fume hood. Always wear a lab coat, gloves and safety glasses.
• Isopropanol (molecular biology grade).
• 75% Ethanol wash (molecular biology grade).
• RNase-free glycogen (catalogue #: 10901393001 [Roche CA, USA]).
• Nuclease-free water (RNase-, DNAse- pyrogen-free) (catalogue #: AM9932 [Life Technologies]).
• Synthetic single-stranded RNA oligonucleotide for use as an exogenous spike-in control. This can be any sequence not found in the host organism. For analysis of human and mouse serum the Caenorhabditis elegans miRNA cel-miR-39 is typically used (5′-UCACCGGGUGUAAAUCAGCUUG). Alternatively a cocktail of synthetic small RNAs can also be used, in which case multiple oligonucleotides are required. (NOTE: 5′ phosphorylation of the RNA oligonucleotide is not required).
• MicroRNA Reverse Transcription Kit (catalogue #: 4366597 [Life Technologies]).
• TaqMan Gene Expression Mastermix (catalogue #: 4369510 [Life Technologies]).
• Small RNA TaqMan assay (medium/large scale) (Life Technologies). (Assay IDs: miR-1 002222, miR-133a 002246, miR-206 000510, cel-miR-39 000200). RT primers are supplied at 20X concentration with medium and large scale assays. This concentration is more convenient for RT multiplexing whereas the small scale assays contain 5X RT primers which is less convenient.
• Yeast tRNA (catalogue #: 10109495001 [Roche]).
• MS2 Bacteriophage RNA (catalogue #: 10165948001 [Roche]).
• TRIzol LS Reagent (catalogue #: 10296-028 [Life Technologies]).
• Chloroform (molecular biology grade)
• Isopropanol (molecular biology grade).
• 75% Ethanol wash (molecular biology grade).
• RNase-free glycogen (catalogue #: 10901393001 [Roche CA, USA]).
• Nuclease-free water (RNase-, DNAse- pyrogen-free) (catalogue #: AM9932 [Life Technologies]).
• Synthetic single-stranded RNA oligonucleotide for use as an exogenous spike-in control. This can be any sequence not found in the host organism. For analysis of human and mouse serum the Caenorhabditis elegans miRNA cel-miR-39 is typically used (5′-UCACCGGGUGUAAAUCAGCUUG). Alternatively a cocktail of synthetic small RNAs can also be used, in which case multiple oligonucleotides are required. (NOTE: 5′ phosphorylation of the RNA oligonucleotide is not required).
• MicroRNA Reverse Transcription Kit (catalogue #: 4366597 [Life Technologies]).
• TaqMan Gene Expression Mastermix (catalogue #: 4369510 [Life Technologies]).
• Small RNA TaqMan assay (medium/large scale) (Life Technologies). (Assay IDs: miR-1 002222, miR-133a 002246, miR-206 000510, cel-miR-39 000200). RT primers are supplied at 20X concentration with medium and large scale assays. This concentration is more convenient for RT multiplexing whereas the small scale assays contain 5X RT primers which is less convenient.
• Yeast tRNA (catalogue #: 10109495001 [Roche]).
• MS2 Bacteriophage RNA (catalogue #: 10165948001 [Roche]).
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