Figure 6. Expression of 16SrRNA-s. RT-PCR with primers that amplify the 2010-3075th region of mtDNA detects a 300-bp band and a 500-bp band, besides the anticipated band at about 1-kb, in HEK293 cells.

The 500-bp band occurs randomly with greatly variable density, thus likely being a heterodimer of the 300-bp and 1-kb cDNAs, which appears randomly and is a common phenomenon as we have frequently described [37], [39], [40]. Isolation of the 300-bp band followed by T-A cloning and sequencing reveals that it is an mtRNA with a 768-bp (the 2069–2836th nt region of mtDNA) deletion, as indicated in the sequence. The boldfaced sequence is the downstream exon. The number of the nt is based on the UCSC browser, with the position of the last nt in the reverse primer (underlined) and the first nt in the forward primer (underlined) indicated. The sequences outside the primers belong to the T-A vector. Because the RNA sample was not pre-digested with DNase, the 1-kb band should be derived from not only cDNA, but also mtDNA, of the 16S rRNA gene. Removal of DNA from an RNA sample from Hela cells followed by RT-PCR (cDNA) still detects the 300-bp band, besides a band at about 850-bp, while the 1-kb band was very weak. Cloning and sequencing the 850-bp band reveal that it is part of the 16S rRNA that was amplified because the reverse primer is also partly reverse-complementary to the mtDNA. Direct PCR amplification of the RNA sample without RT (RNA) detects only the 1-kb band, but not the 300-bp one, indicating that the 300-bp band is not associated with an mtDNA with deletion.