Table 4.

Advantages and limitations of different approaches for mitochondrial RNA expression analysis

RNA expression analysis
Method	Advantages	Limitations
next-generation sequencing	✓high throughput ✓identification of novel ncRNAs and their modifications	✕ laborious and long bioinformatic analysis ✕ bias introduced during library preparation
single-molecule real-time sequencing/Nanopore sequencing	✓generation of full-length transcripts ✓identification of novel ncRNAs and their modifications ✓amplification bias bypassed	✕ laborious and long bioinformatic analysis ✕ high cost
qPCR	✓rapid ✓cost-effective ✓absolute (standard curve required) and relative quantification	✕ limited to known ncRNAs ✕ lack of reliable reference RNAs ✕ sensitive to RNA quality
droplet digital PCR	✓no standard curve required for absolute quantification ✓discriminatory power for low-input or low-quality samples	✕ limited to known ncRNAs ✕ low dynamic range compared with qPCR ✕ high cost