Article added to library!
x
Pubchase is a service of protocols.io - free, open access, crowdsourced protocols repository. Explore protocols.
Sign in
Reset password
or connect with
Facebook
By signing in you are agreeing to our
Terms Of Service and Privacy Policy

ESSAY ON PAPER

Visualizing SNVs to quantify allele-specific expression in single cells.
Aug 29, 2013   Nature methods
Levesque MJ, Ginart P, Wei Y, Raj A

COMMENTS

What is PubChase?

Read the essays and get personalized biomed recommendations on your mobile device

4

An "overnight" success

Aug 13, 2013
1707 views
Sometimes an idea just works. It's SOOO satisfying when that happens! Whenever it does, I almost feel like it was luck or cheating. But then I think about it a little bit more, and usually it turns out the ideas and the execution have their origins in a deep knowledge acquired through years of work. That's totally what happened with our SNP FISH paper.

Our paper is really simple. Basically, we describe a method that allows us to discriminate single base changes on individual RNA molecules through fluorescence in situ hybridization. It's totally science fiction! But it basically boils down to a couple simple tricks. First, you use a "mask" on your detection probe to block off part of the sequence to increase probe specificity. Then, you use co-localization with "guide" probes that locate the target RNA precisely, allowing one to easily discriminate legit binding from non-specific binding. Sounds simple!

The history of this project is that Marshall (who's a real gear head when it comes to technical stuff) was very interested in coming up with a way to reduce background in our conventional single molecule RNA FISH. He had tried all kinds of things over the span of a couple years, and in the course of doing so, had been working with mask oligonucleotides. He developed all sorts of algorithms for probe design and tried out many different combinations of oligo designs, but in the end, we realized that we needed some sort of way to quickly and effectively test oligo hybridization. At some point, we realized that the best way to do this was to watch a single oligo bind and measure its hybridization efficiency. And this worked, which was great (although it required endless messing around with various dye chemistries, yuck!). So Marshall kept plugging away at that for a spell, when one day he came in, came up to the whiteboard, and said something like "Check this out, I think we can do SNP detection...". It was a really cool moment, because it was one of those rare instances when you have an idea and you just know it will work deep down in your gut. It was also a really special moment for me as a PI, because it was Marshall's idea, and I was so very proud of him for coming up with it.

And work it did, pretty much right away. We basically verified that the principle was solid within something like a couple weeks, then spent the next several months doing the usual prettification that you need for a glossy paper (with huge statistical help from Paul Ginart, who is carrying the SNP FISH torch forward in the lab). But while it seemed to come together so quickly, when I think back on it, it was all of Marshall's years of super technical work with mask probes and dye combinations and so forth that made it happen. I guess the lesson is that you have to work hard all your life to become an overnight success.
Copyright: © 2013 Arjun Raj. The above content is licensed under the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.  
If you find this essay offensive or in violation of your rights, please email to pubchase@zappylab.com
x

Find my publications in PubMed

We will use the information below to search for your articles.
*
x

Essay Drafts