MicroRNA (miRNA) in Human Mitochondria

Our bioanalytical paper of the month May 2011 is published by Barrey E et al (2011): Pre-microRNA and mature microRNA in human mitochondria. PLoS ONE 6(5), e20220. The authors used supramagnetic microbeads for the isolation of intact mitochondria from human tissue. The paper demonstrates the power of immuno-beads for the capture of virtually any ligand from the liquid phase.
Another post which might be of interest to you is about antagomirs.
Best wishes, your binding-assay.com team.

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The Proximity Ligation Assay – Proximal to Ultimative Assay Sensitivity?

yesterday we had the Ascension Day, also called the Father´s Day in Germany, which means all fathers enjoyed the day along with their kids outside under the fantastic blue sky! And that´s what I did, and got a slight sunborn on my nose …
Back in the office this morning I came across the fantastic proximity ligation assay, possibly an even more sensitive alternative to the immuno qPCR assay. Both immunoassays have superior sensitivity in comparison to the classic ELISAs (ancient, I´d better say).

If you review all the ligand binding assays we´ve posted so far, you must admit all innovative therapeutics currently in the Biotech´s pipelines (peptides, antibodies, proteins, siRNAs, microRNAs, miRNAs, etc) can be analyzed with only one device: a Real-Time RT-qPCR system fully compatible to Good Laboratory Practice (GLP). It´s good to see there´s at least one

Enjoy your weekend,
Your binding-assay.com team!

 

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The Invention of Immuno RT-qPCR is in Stand-By Position

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

 

References:
(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.

PyroPhage® is a registered trademark owned by Lucigen Corp.

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The SELEX Assay – Beyond The Right Track

Why has the systematic evolution of ligands by exponential enrichment (SELEX) become so unpopular? What is the reason why everybody puts much more resources on peptide and antibody based drugs rather than small RNA or small DNA aptamers?

We strongly believe there´s a way back on the right track. The Altermune project shows in the right direction.

All the best for a lovely weekend, your binding-assay.com team.

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Immuno qPCR Where Other Assays Flop

Dear all,
after some time of relaxing, we are now proud to provide another exciting ligand binding assay: the immuno qPCR as ultra-sensitive tool for protein or peptide ligand detection. This assay comes both in the microplate and magnetic bead assay format.
Have fun, your binding-assay.com team.

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