Quantitative Analysis of Mitochondrial tRNAs

Total mitochondrial RNAs (mtRNAs) were obtained from mitochondria isolated from mutant and control cell lines (∼2.0 × 10⁸ cells) using a ToTALLY RNA kit (Ambion), as described previously (31 (link)). Two micrograms of total mitochondrial RNA was electrophoresed through a 10% polyacrylamide–7 M urea gel in Tris-borate-EDTA (TBE) buffer (after heating the sample at 65°C for 10 min) and then electroblotted onto a positively charged nylon membrane (Roche) for hybridization analysis with oligodeoxynucleotide probes. For the detection of tRNA^Ala, tRNA^Lys, tRNA^His, and tRNA^Glu, nonradioactive digoxigenin (DIG)-labeled oligodeoxynucleotides specific to each RNA and 5S RNA were as detailed previously (9 (link), 15 (link), 32 (link)– (link)34 (link)). DIG-labeled oligodeoxynucleotides were generated by using a DIG oligonucleotide tailing kit (Roche). The hybridization was carried out as detailed elsewhere (9 (link), 15 (link), 32 (link)– (link)34 (link)). Quantification of density in each band was done as detailed previously (9 (link), 15 (link), 32 (link)– (link)34 (link)).

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Jiang P., Wang M., Xue L., Xiao Y., Yu J., Wang H., Yao J., Liu H., Peng Y., Liu H., Li H., Chen Y, & Guan M.X. (2016). A Hypertension-Associated tRNAAla Mutation Alters tRNA Metabolism and Mitochondrial Function. Molecular and Cellular Biology, 36(14), 1920-1930.

Publication 2016

ToTALLY RNA kit positively charged nylon membrane DIG oligonucleotide tailing kit

Cell lines Cells Digoxigenin Hybridization Membrane Mitochondria Mitochondrial rna Nylon Oligodeoxynucleotides Oligonucleotide Polyacrylamide gel Tris borate edta buffer Trnaala Trnaglu Trnahis Trnalys Urea

Corresponding Organization :

Other organizations : Zhejiang University, Wenzhou Medical University, First Affiliated Hospital of Wenzhou Medical University

Top 5 similar protocols

Variable analysis

independent variables

Mutant cell lines
Control cell lines

dependent variables

Total mitochondrial RNAs (mtRNAs)

control variables

Cell count (approximately 2.0 × 10^8 cells)
RNA extraction method (ToTALLY RNA kit)
Gel electrophoresis (10% polyacrylamide–7 M urea gel in Tris-borate-EDTA (TBE) buffer)
Blotting method (Electroblotted onto a positively charged nylon membrane)
Probe detection (Nonradioactive digoxigenin (DIG)-labeled oligodeoxynucleotides for tRNA^Ala, tRNA^Lys, tRNA^His, tRNA^Glu, and 5S RNA)

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