Real-Time RT-qPCR

4. Almost No Influence of Small RNA 2´-O-Methyl Modifications

Merck & Co scientists published a basic research paper on the Real-Time RT-qPCR assay selection and development. They found the stem-loop primer mediated reverse transcription quite useful to quantify 2´-O-methyl modified guide (antisense) strand of a ds HBV 263 siRNA (1). Upon anealing of the stem-loop RT primer, not less than seven 2´-O-methyl modified Ribonucleotides can be transcribed into DNA. Applied Biosystem´s Multiscribe RT was applied, which is a M-MuLV RT derivate. Remember the guide strand is the molecule which guides Argonaute (Ago) endonucleases to the target RNA.

Another Merck & Co paper clearly demonstrated the ability of M-MuLV RT derivatives to reverse transcribe Ssb targeting ds siRNA bearing up to eight 2´-O-methyl modifications (2). To our knowledge so far, this is the first publication which describes the combination of the sensitive Real-Time RT-qPCR liquid phase assay with previous Argonaute (Ago2) immunoprecipitation – incredibly pushing the assay sensitivity to homoeopathic nucleic acid drug dose levels.

Alnylam scientists used the same commercially available kit (as Merck & Co did) comprising a M-MuLV reverse transcriptase to quantify 2´-O-methyl siRNA in plasma and liver lysates – without prior extraction of the RNA (3). According to the paper, the M-MuLV reverse transcriptase managed to transcribe the A5475 guide (antisense) strand of a ds siRNA targeting human ApoB (4), though this strand bears five 2´-O-methyl modifications.

Scientists of New England Biolabs (NEB) reported some difficulties in the reverse transcription of piwi-interacting RNA (piRNA), which usually bear a single terminal 2´-O-methyl modification at its 3´-end (5). Probably NEB tried to develop a piRNA cloning kit for next generation sequencing techniques (quite similar to Illumina´s T4 RNA Ligase 2 approach (6), in my opinion one of the last decade´s innovations). But the 2´-O-methyl remains challenging.

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