Amplicons of a ND5 mRNA segment to detect m1A1374 levels in HEK APPwt and Ctl cells
This experiment was set up to quantify m1A methylation levels at position 1374 of the mitochondrial encoded ND5 mRNA. To investigate the relevance of this methylation in Alzheimer's disease two cell lines were compared. HEK APPwt cells, transfected with a plasmid encoding the wild-type Amyloid-Precursor-protein and untransfected control cells were used.  In the first step a RT was performed with SuperScript-IV (SS-IV) and a ND5 specific RT primer. Afterwards a PCR was conducted to amplify the sequence around the m1A position. The amplicon is about 100 bp length and was sequenced in an Illumina Sequencing was performed in a MiSeq 2x75 PE run. For both cell lines the m1A analysis method was conducted with 4 biological replicates of total RNA and 2 biological replicates of RNA isolated from mitochondrial extracts.