Open AccessThe mitochondrial housekeeping gene 16S is inappropriate as an internal standard in comparative studies of rare mitochondrial transcripts using S1-nuclease protection assays
Author(s) -
Sandra Ebert,
Line Breumlund,
Roland Nau,
Uwe Michel
Publication year - 2010
Publication title -
journal of nucleic acids investigation
Language(s) - English
Resource type - Journals
eISSN - 2035-6005
pISSN - 2036-7996
DOI - 10.4081/jnai.2010.e2
Subject(s) - nuclease , biology , mitochondrial dna , microbiology and biotechnology , rnase p , housekeeping gene , nuclease protection assay , rna , ribosomal rna , gene , gene expression , genetics , non coding rna
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