We've performed Profiling of Ago-2 RIP miRNA in tissue and also followed it with individual RT-qPCRs and showed that the miRNA levels between the 2 were correlated but this is with a handul of miRNa species... has there been intensive studying the relationship between miRNA present in the cell and the amount that is loaded
Hello Aidan.
As you know, none exists. This is probably due to the complexities lying the processing from ncRNA sequences, pre-miRNA, pri-miRNA and mature miRNA, such a bundle of species! In addition, their spatial expression or working are not completely known, showing in the nucleus, cytoplasm, extracellular space in the vicinity of generating cells (as form of exosomes), and long-range body fluids.
If someone make distinct transgenic ncRNA cells or animals (there is a few reports), then it would be very useful model to determine such a profile of spatio-temporal expression or fates of the TG ncRNA within the cell or something else.
Regards.
Hi Aiden,
There are few studies that say that the amount of total miRNA is usually in excess compared to the total argonaute molecules. Janas et al., reported that the total amount of miRNA in a cell was 13 fold in excess compared to the total argonaute protein in mammalian cells. Out of the total argonaute-associated-miRNAs, only a fraction is actively involved in miRNA-mediated gene silencing (Rocca et al., 2015).
Janas et al: (DOI: 10.1261/rna.035675.112)
http://rnajournal.cshlp.org/content/18/11/2041
Rocca et al: (DOI: 10.1073/pnas.1424217112)
http://www.pnas.org/content/112/3/767
The correlation you see with AGO2-RNA immunoprecipitation (RIP) and individual RT-qPCR could be because of how the general RT-qPCR works.
The general protocol for RT-qPCR is optimized to detect only the mature microRNAs (single stranded miRNAs released from the argonaute during RNA extraction). It does not detect the levels of duplex miRNA (argonaute free miRNA) unless a denaturation and primer annealing step is performed before the reverse transcriptase (RT) reaction.
If you have done the RT-qPCR to detect both single and double stranded miRNAs, then the correlation what you see is real. With increasing cellular abundance, there is more chance of the miRNA being loaded on to the RISC complex (Janas et al., 2012).
Best wishes.
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