RT in this context means “reverse transcription”, not “real-time” as recently suggested (1). When I skimmed through the immuno qPCR publications on Pubmed, I was wondering a bit why nobody have been dealing with reverse-transcription to further increase the sensitivity of the immuno qPCR assay. Especially in the context of the massive competitive pressure from Olink Bioscience/Invitrogen, who push the proximity ligation immunoassay technique into the diagnostics market.
Usually a double-stranded DNA tag is covalently attached to an antibody. For instance, a 563-bp fragment of firefly (Photinus pyralis) luciferase is coupled to generate a 101 bp internal region fragment as amplicon (1). In theory, this ensures that only one template of a DNA tag produces one copy in the first cycle of the qPCR assay. It is obvious that more template molecules will produce more copies during first qPCR assay cycles, which should promote an even more sentitive qPCR assay.
One approach to improve qPCR assay sensitivity could be the substitution of the DNA tag by a single-stranded, chemically stabilized RNA. In the latter example, a 563 nt single-stranded RNA would produce – if reverse transcribed by using random hexamers, approx. 70-90 cDNA copies per RNA template. The amplicon region should be close to the RNA´s 5´-end.
Another approach could be a single-stranded small RNA (e.g. like a siRNA, microRNA, miRNA) tagged to the antibody. If reverse transcriped by the commonly used hairpin primers in combination with a M-MuLV reverse transcriptase, the background amplification signal should be reduced to almost zero (because the RNA can be heat-degraded). If you use the RNase H+ wildtype RT, the RNA is destroyed enzymatically. The introduction of a thermostable reverse transcriptase (e.g. like Lucigen´s PyroPhage® RT), opens the possibility to develop a one-step immuno RT-qPCR assay.
And while we´re at it, what about the RCA assay to amplify an RNA template tag before the reverse transcription?
And what if you use a single-stranded small RNA tags as a template for a RNA dependent RNA polymerase (2) to generate the double-strand? If you use the new generated small RNA single strand for a one-step hairpin-primed RT-qPCR assay, the background should also be reduced to almost zero, shouldn´t it?
What do you think? Let us know your opinion!
Have a nice weekend,
Your binding-assay.com team
(1) Babu D & Muriana PM (2011), J Microbiol Methods, doi:10.1016/j.mimet.2011.05.002
(2) Rohayem J et al (2006), J Gen Virol 87(9): 2621-30.
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