For genomic DNA sequencing, cells were lyzed in lysis buffer (50 mM Tris, pH = 8.0, 20 mM NaCl, 1 mM EDTA, 1% SDS) with proteinase K. DNA was extracted using phenol-chloroform isoamylalcool (ref. P2069, Sigma) and isopropanol (ref. 59309-1L, Sigma). For cDNA sequencing, RNA was extracted using the miRNA Micro Kit (QIAGEN) and reverse transcription was performed using the SuperScript ® VILO cDNA Synthesis Kit (ref. 11754–250, Invitrogen). The region encompassing the mutation was amplified by PCR using 5 or 4 sets of primers for the genomic DNA and cDNA, respectively (Supplementary Table S1 ) and the Kapa2G polymerase (KAPA 2G Robust HotStart PCR Kits, ref. KK5517, Clinisciences). PCR products were run on agarose gels and purified using phosphatase (FastAP, ref. EF0651, ThermoScientific) and exonuclease enzymes (Exonuclease I, EN0581, ThermoScientific). Controls were performed in the absence of reverse transcriptase. Sequencing was performed using the BigDye®TerminatorV3.1 cycle sequencing and purifications kits (ref. 4337455,4376484, ThermoFisher).
Mirna micro kit
Manufactured by Qiagen
Sourced in United States, Germany
The MiRNA Micro Kit is a laboratory equipment product from Qiagen designed for the isolation and purification of microRNA (miRNA) from various sample types. The kit utilizes a silica-membrane-based technology to capture and extract miRNA molecules from the samples.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Exonuclease 1, by Thermo Fisher Scientific (1 mentions)
Superscript vilo cdna synthesis kit, by Qiagen (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Glycogen, by Qiagen (1 mentions)
Bioanalyzer rna pico kit, by Agilent Technologies (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Qiazol, by Qiagen (1 mentions)
Tissueruptor, by Qiagen (1 mentions)
Ovation picosl wta system v2, by Tecan (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
Bioanalyser, by Agilent Technologies (1 mentions)
5 protocols using mirna micro kit
1
DNA and cDNA Sequencing Protocol
2
Total RNA Isolation from Minced Tendon
3
miRNA Isolation and Amplification
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RNA isolation was performed using the miRNA micro kit (Qiagen) according to the manual. On a column, DNase I treatment was applied to degrade DNA. 14 μl of nuclease-free water were added to elute the RNA fractions, which were used immediately or stored at −80 °C. Due to the low amounts of RNA, RNA amplification was carried out using the Ovation® PicoSL WTA SystemV2 (Nugen). The amplification process was done according to the manual using 50 ng total RNA.
4
Hippocampal RNA Extraction and Sequencing
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RNA extraction from dorsal hippocampus was performed using the RNeasy Lipid Tissue Mini Kit (Qiagen). Total cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, USA). For reverse transcription, it was used 200 ng of total RNA from each sample. A solution-phase assay was carried out (Applied Biosystems). Small RNA extraction was done using the miRNA Micro Kit (Qiagen), and the checking of RNA purity and integrity was performed with NanoDrop (Thermo Fisher) and Bioanalyzer (Agilent), respectively. The preparation of the small RNA library required several enzymatic steps to include in the final library only the small RNA fragments. Sequencing of multiplexed libraries was performed in the NextSeq 500 equipment (Illumina).
5
Transcriptome Analysis of Muscle Biopsies
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Total RNA was extracted from 5–10 mg of muscle biopsies from the same participants described above using miRNA micro kit (Qiagen, Hilden, Germany) as per the manufacturer’s protocol, with additional DNase treatment. All RNA samples with RNA integrity number RIN ≥7 (Bioanalyser, Agilent Technologies, Germany), were selected for microarray analysis. Transcriptome analysis was carried out by Oaklabs (Berlin, Germany) on their experimentally validated ArrayXS Human (design ID 079407, Agilent 60-mer SurePrint technology, Agilent Technologies). Gene ontology analysis was performed using David data base tools71 (link), with cutoff enrichment score set above 1.7 and enriched p value < 0.05 (Fisher test).
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