Nanostring Scientists Reveal the “Secret Sauce” in Lysate Protocol Development

qwalton on May 29, 2019

My name is Chris Merritt. I am a Senior Scientist at NanoString Technologies and work in the R&D division. I’ve been here for five years now.

There was one very specific change we had to make to this lysate protocol to make it compatible with the nCounter® platform, and this was the addition of a proteinase K stub. We found out that the gunk from crude lysate interfered with the B-purification that’s very specific to NanoString and that stub addition really helped boost counts in the end for this assay.

This updated protocol, it’s very similar to the previous protocol; you will get very similar results using this. We updated and gave more details on the different steps that the user must do for the protocol, and we ran it with many other users in the lab. It showed highly reproducible and works across many cell lines. Previously we had only looked at a few cell lines.

The second aspect of this assay is that we had to develop three specific protocols with three different types of cells. We have a specific protocol for suspension cell lines, a protocol for adherent cell lines, and the final protocol is for primary cells for both human or mouse cells or basically any primary cell type that one would find in a lab.

We focused on three broad classes of cell lines to look at: suspension cell lines, adherent cell lines and primary cells. At first, we looked at the commonly used cell lines like Hela cells, A431, or Jurkat cells. We then moved on and expanded the list of cells to about 20 different cell lines or cell types to make sure it’s compatible with all of these. The last thing we did for the protocol is to make sure it was compatible with a 96-well format, because this is very important when you’re trying to do higher throughput assays, which we’re trying to do with the PlexSet™ assay.

When I was in academia, I would develop new protocols for the experiment I was working on, or that very specific assay I was trying to figure out. If it worked out, if it’s a novel assay, I would need to teach others how to run it, so they could also apply it to their experiments. Or I would work with neighboring labs to work on a similar system and teach them. NanoString is a large company and we have users across the world. I, again, don’t have the luxury to interact with every customer and help teach them how to run the protocol, so we need to make sure the protocol is very reproducible and very clear and will basically work in anyone’s hands with just a printout of the protocol. When we’re doing validation, we look across different types of users. We have super users in the lab who’ve run their protocol and help develop their protocol, which for them, the protocol’s going to typically work quite well. What we want to do after that is bring in some naive users to make sure it works in the hands of others.

In this case, we took two naive users and one experienced user. Basically, we found that the data looked quite similar and got nice correlation for purified RNA and for lysates. When we’re developing a new protocol, like this new lysate protocol or even just a new product, the first thing that happens at a company like NanoString, is Marketing will go out there and figure out the voice of the customer, what they’re looking for. Then they’ll work closely with R&D, with a Scientist like me, to figure out what are the requirements for those products to make sure it’s going to meet the need of the customer.

Once we figure that out, we go through right specifications for the product and eventually write down protocols that we’re going to test to make sure the protocol or assay will work in their hands. This is a rigorous process. Once we get the validation data, we meet as a group in R&D and at different parts of the company, make sure it passed all the criteria we laid out early on, and then eventually we start interacting with early customers, which we’ll call beta customers sometimes, to make sure it works in their hands. After that, we’ll train internal NanoString folks, like the Field Application Scientists or the Tech Services team; we’ll teach them in the lab in Seattle, how to run the assays so they can go out there or when they’re on the phone with customers, to be educated in how to run the assay.

When we did the validation of this new protocol, the first thing we did is to make sure we were getting linear results in terms of, if you add more lysate, do I get more counts? Which you do for this assay. The next part of the validation was to make sure it worked across all different cell types you might see in the lab. We can’t test all cell lines at NanoString- that’s just too much effort. We found three buckets that we could look at, these are adherent cell lines, suspension cell lines and primary cells. We looked across all these three different cell types. These are examples of forehand cell lines, four suspension cell lines and four primary cell types across human and mice showing that purified RNA data looks very similar to data obtained with crude lysates.

In the end, we try to make it easier for the customer to analyze the nCounter counts when they come out of the system. For that we developed the nSolver™ software packages and advanced analysis pack. We developed this new lysate assay for PlexSet specifically, but we found that it works for all nCounter Gene Expression Assays. What we’ve shown in the validation is it worked quite well user-to-user, and we’re excited for people to use it out in the wild because we think it’s going to work quite well, and it’s quite efficient in getting their data much faster.

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

Post by qwalton