FAQ explaining why MagIC beads offer a large number of advantages over competing products on the market
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Q: For the SARS-CoV2 RNA pulldown for NGS – how do investigators know if there is a new mutation in the target sequence or not that causes the pulldown to be less efficient than normal – clearly not an issue for standard mRNAs and DNAs but with any rapidly mutating viral RNAs, particularly HIV for example – are there any work arounds or ways to check what has been pulled down and what has not in one’s samples?
A: This is an important issue in such studies. There are no methods, which can assay the potential issues caused by viral mutations prior to sequencing them. There are also no smart or cost-effective ways to check what has been pulled down and what has not in one's sample.
The issue in question is far more problematic when using products like the NGS kits from similar approaches sold by other suppliers and is less of an issue with Element Zero MagIC beads. See our handy guide here: PDF LINK
The mutations of the viral sequences tend to be quite localized whenever they happen. Therefore there are only parts of the viral sequence, which can change significantly enough to not allow the capture probe to hybridize to them. When strong sequence changes occur and one is using NGS kits from other suppliers the mutated regions will end up not being captured and will be missing in the output of the sequencing run. This is because the technology from other companies (unlike MagIC beads) is designed to capture the target sequences after they have been heavily fragmented and the capture of each of the fragments of the viral RNA has to be directly hybridized to the capture probes. The mutated region will no longer be complementary to the capture probe and will end up being omitted in the enrichment whereas all of the non-mutated parts of the sequence will be captured without a problem resulting in a defined gap in the output of the sequencing run.
MagIC Beads have a clear advantage here as in contrast to old school enrichments in that MagIC beads are designed to capture unfragmented RNAs. This means that although the mutations can also prevent the hybridization of a capture probe to target sequence this will not translate into a loss of a given segment of a sequence in the enrichment. For example, when a given RNA molecule is targeted with 10 MagIC bead capture probes the unsuccessful hybridization of even a half of them will still result in the efficient capture of the sequence as the neighboring capture probes will circumvent for the loss of the binding sites of other capture probes.
Q: Can you explain to me what such good enrichment relative to really abundant RNAs like ribosomal RNAs and GAPDH means?
A: The enrichment relative to non-target RNAs tells how many times the level of the given non-target RNA has been reduced in relation to the target in enriched sample when compared to the starting material. It is a basic metric of the efficiency of sequence specific enrichment of nucleic acids and is generally accepted as a proof of the enrichment efficiency.
In practical terms if in the starting sample the levels of GAPDH are 1000x higher than that of the RNA of interest whenever the sample will be analyzed by sequencing one can expect to generate the sequencing reads prevalently from GAPDH and other abundant RNAs leading to a very tiny fraction of the % of the reads coming from the RNA of interest. The enrichment of the target to the abundant RNAs informs on how much these dynamics have changed and how much the complexity of the sample has been reduced, which translates to how focused the downstream analysis done after the enrichment will be on the molecule of interest.
Q: What would the data look like if you compared the fold enrichment relative to a control RNA that has similar abundance to the target RNA in cells?
A: It would look similarly to the enrichment over the abundant RNAs. We do have the data, which proves this, but the enrichment over not abundant RNAs is much less valuable than over the abundant ones. The aim of the enrichment is to reduce the complexity of the sample and bring the focus of the downstream analysis to the molecule of interest. Non-targets, which are not abundant do not increase the complexity of the sample much and do not dilute the focus of the downstream analysis as much as the abundant ones do. Therefore, the efficiency shown over the abundant non-targets is more important to display.
RNA Interactome pull-downs
Q: Are you concerned about the authenticity of the identified interactors of the target RNA in the interactome studies? E.g. if a subset of proteins is identified in an experiment investigating the interactome of an RNA of interest how can one be sure that the identified proteins are the true interactors of the RNA and not just non-specifically recovered proteins.
A1: We do not think any widely accepted metrics exist to address this as the problem is potentially very variable between the types of samples investigated. Classically the way to deal with the issue is to use controls. A typical control in this setup would be using the beads specific for the target of interest side by side with the beads, which are supposed to be non-targeting for any RNA in the sample. When interactors recovered from both beads are identified one can use what has been pulled down with the negative control beads as a representation of the experimental background. In an ideal world, what has been captured with non-targeting beads should define the experimental background, which can be then inferred from the results obtained from the targeting beads. The proteins uniquely recovered with the targeting beads should represent the true interactors. This kind of control, however, often does not work so well and people have mostly moved away from using it.
A2: For academic research, the only truly reliable way to control the issue (the one that reviewers of scientific publications request in the process of publishing) is a series of reverse experiments. The principal is simple enough. If you have co-captured proteins alongside your RNA you might have to show that you can co-capture the same RNA when pulling down the proteins in question. When you know for which RNA-protein interaction to test those reverse experiments from the protein perspective are very straightforward.