Our protocol is a concise 2-day RNA FISH protocol. The goal of FISH is to detect the existence and/or quantify the abundance of an RNA of interest in the whole-mount adult fly brain. Our protocol is adapted from the one originally reported in https://doi.org/10.1016/j.ymeth.2017.06.025.

Probe design#

Our probes come from a company named Biosearch Technology. The principle of their probes is: a library of 50 oligos will be used to hybridize onto your RNA. Each oligo is 18-22nt long. Only when most of the 50 oligos bind to the RNA molecule will the fluorescence shine. This technology has greatly improved the SNR.

There are two ways to get your FISH probes:

  1. Search existing probe for your RNA of interest

    This method does not work all the time since previous papers may not have developed probes for your RNA of interest or they may use a different probe manufacturer other than Biosearch Tech. But you should always search first, because testing a new probe is very time-consuming.

    Here are two papers that report tested probes for many neurotransmitter identity-related RNAs: https://doi.org/10.1534/genetics.118.301749; https://doi-org.ezp-prod1.hul.harvard.edu/10.1038/nmeth.4309.

  2. Design customized probes using Stellaris designer tool

    Go to https://www.biosearchtech.com/support/tools/design-software/stellaris-probe-designer. Click “start design”. In the design page, you can first leave all the parameters default.

    Input the target sequence, which can be found on FlyBase database (specifically, under each gene’s entry, go to “Gene Model and Products –> Transcript Data”. If there are multiple variants, you need to firstly determine either all variants or selected variant(s) you intend to detect. For detection of multiple variants, the input sequence should be the sequence that is common to all variants, which may require you to use bioinformatic sequence alignment tool. In contrast, for single-variant detection, the input sequence must be void of the sequence that is common to other transcript variants)

    Then run the designer and you will get your probe design in minutes. With the designed probe library, you can proceed to order your probe! In the situation that your library has less than 48 oligos, refer to their probe design blog (https://blog.biosearchtech.com/considerations-for-optimizing-stellaris-rna-fish-probe-design?_gl=1oz5lv7_gcl_auMTk3MTE1NzQwMS4xNjkyMTE2MzY3_gaNTg3NDUwMjg0LjE2OTIxMTYzNjc._ga_QLQ6ZQ021Q*MTY5OTM3MjA0Ny4xMi4xLjE2OTkzNzIyNTIuNjAuMC4w) and follow the instructions to play with the parameters and run the designer again, until you get enough oligos in your probe library.

In either case, you finally need to choose a fluorephore for your probe. As recommended by Biosearch Tech, their best are Quasar® 570, and then Quasar® 670. If you need to multiplex more than 2 RNAs, you might want to consult Biosearch Tech which fluorephores to use next.

FISH protocol#

📎 FISH protocol-11.4.2023.docx

  • Chennan has been using 0.2mL tubes for FISH because they fit into the PCR thermal machine. We need to use this machine because it gives easy, programmable, and accurate control of temperature. 0.2mL tubes for FISH are labeled “FISH only” in the molecular biology drawer.
  • All the reagents for FISH need to be dissolved with nuclease-free water, no exception for PFA. They are stored in the “FISH reagents” box above the back bench. Some reagents (e.g. Formamide) are stored in -20C freezer in the front lab in a box labeled “RNA FISH”.
  • It’s good practice to frequently spray and swipe the bench surface with RNaseZap in order to protect your RNA from degradation. Wenyi has also suggested not talking during FISH process.

How to use the PCR T100 Thermal Cycler#

  • Select a protocol: There is already a thermal protocol called “FISH” saved in the machine. This protocol is for FISH tubes with 50uL solution inside. The temperature is set at 37C for infinite length of time, so that you can feel free to take your samples out whatever your desired duration of time is. Alternatively, you can create your own protocol by following the instructions on the User’s Manual card next to the machine.
  • Steps to start: First turn on the switch at the back. Then select/make a protocol and click “run”. You need to wait a bit until the temperature ramps up to your desired temperature. Then put your samples in. When you finish, make sure to turn off the switch.

Tips on troubleshooting/optimizing when FISH is not working#

Typically this happens when you got a new probe. If you don’t see any FISH signal (FN) or your FISH signal is everywhere (FP) following the regular FISH protocol, there’re several parameters that you may consider varying:

  1. probe concentration

  2. hybridization temperature

     lower temperature gives less specificity but more sensitivity; higher temperature gives more specificity but less sensitivity (specific means that the probe binds to your target RNA but not other random RNAs; sensitive means that the probe effectively binds to most of your target RNA and thus you get bright signal). 

 You could try lowering your temperature if you don't see any signal (FN); and raising your temperature if you see too much signal (FP). 

 Testing multiple temperatures simultaneously can be done on the PCR machine by using the "gradient" function to program different temperature for each row.
    1. wash duration

If all these still don’t fix your problem, consult Wenyi or search online for FISH troubleshooting blogs.