The mitochondrial housekeeping gene 16S is inappropriate as an internal standard in comparative studies of rare mitochondrial transcripts using S1-nuclease protection assays

  • Sandra Ebert | sebert1@gwdg.de University of Göttingen, Department of Neurology, Germany.
  • Line Breumlund Universtity of Göttingen, Department of Neurology, Germany.
  • Roland Nau University of Göttingen, Department of Neurology, Germany.
  • Uwe Michel University of Göttingen, Department of Neurology, Germany.

Abstract

The analysis of rare mitochondrial transcripts derived from the L-strand of the mitochondrial genome requires a sensitive method such as the S1-nuclease protection assay. We examined whether the ribosomal mitochon­drial transcript 16S is suitable as an internal standard in a multiplex S1-nuclease protection assay for the measurement of different mitochondrial transcripts. For reliable quantification of rare mitochondrial transcripts with the RNase protection assay, a minimum of 2 μg of total RNA is necessary. Standard curves of 16S RNA produced with total RNA from human kidney, liver, brain, and a human neuroblastoma cell line (SH-SY5Y) revealed dose-response relationships that were saturated already at less than 0.5 μg of total RNA. Therefore, 16S is inappropriate as an internal standard for analyzing mitochondrial transcripts with RNase protection assays when more than 0.5 μg of total RNA have to be analyzed.

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Published
2010-04-14
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Keywords:
Rare mitochondrial transcripts, 16S, Housekeeping gene, Internal standard, S1-nuclease protection assay
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How to Cite
Ebert, S., Breumlund, L., Nau, R., & Michel, U. (2010). The mitochondrial housekeeping gene 16S is inappropriate as an internal standard in comparative studies of rare mitochondrial transcripts using S1-nuclease protection assays. Journal of Nucleic Acids Investigation, 1(1), e2. https://doi.org/10.4081/jnai.2010.1588