Biochemistry
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Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH
Chapters
Summary September 20th, 2020
Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.
Transcript
SM FISH accurately quantifies levels and localization of specific RNase. This method is particularly useful for analyzing cell to cell and allele by allele variation in RNA. Gene transcription is an essential process in cell biology, SM FISH allows for the investigation of RNA content in response to external stimuli at the single cell level and provide spatial information.
Following estrogen receptor agonist treatment, wash the cells with cold sterile PBS with calcium and magnesium and fix the cells with 500 microliters of fixation buffer for 30 minutes on ice. At the end of the incubation, wash the cells two times with cold sterile PBS with calcium and magnesium for three minutes before adding 500 microliters of 70%ethanol to each well. Then seal the 24 well plate with a paraffin plastic film and place the plate on a rotator at four degrees Celsius for at least four and up to 16 hours.
For RNA FISH hybridization, at the end of the incubation wash the cells one time with 500 microliters of 2X SSC buffer supplemented with 10%formamide for five minutes at room temperature on a rotator. During the incubation, place a piece of parffin plastic film unexposed side up into a glass Petri dish cleaned with an RNA surface decontaminate and place to sterile water-saturated paper towels along the edges of the dish. At the end of the incubation, place evenly spaced 30 microliter droplets of freshly prepared to GREB1 intron and GREB1 exon probe buffer onto the clean side of the paraffin plastic film and use sterile forceps to gently place one cover slip of cells cell side down onto each droplet without bubbles.
After sealing the dish with paraffin plastic film, cover the plate with foil and incubate the plate overnight at 37 degrees Celsius on a flat non-rotating surface. The next morning, use forceps to return the cover slips to the 24 well plate cell side up for two 15 minute washes in 500 microliters of fresh 2X SSC buffer supplemented with 10%formamide per wash at 37 degrees Celsius on a heated rotator. After the second wash, counterstain the DNA with one microgram per milliliter of DAPI in 2X SSC buffer for 10 minutes at room temperature on a rotator followed by a five minute wash in 2X SSC buffer at room temperature.
Use a paper towel to remove the excess 2X SSC buffer and rinse the samples in nuclease-free water to eliminate excess salts. Then use non-hardening mounting medium to mount the cover slips onto glass slides and seal the cover slips with clear nail polish. For sample imaging, load the slide with the highest expected intensity onto a widefield epifluorescence microscope stage equipped with a 60 or 100 times oil objective in an sCOMS camera and place a droplet of immersion oil onto the objective.
For Zstack acquisition, focus on the sample in the DAPI channel and select the top and bottom of the Zstack at the distances at which the DAPI-stained nuclei become out of focus. Use the slide to optimize the amount of illumination from the light source and exposure times for each channel and acquire images from all of the slides under conditions that avoid photo bleaching and or camera pixel saturation. After the acquisition, use 10 cycles of an aggressive restorative algorithm to deconvolve the images and save all of the projection images according to the bit depth of the camera used.
For image analysis, first download the Jupiter Notebook from GitHub and install Python Anaconda Distribution version 3.6 or higher. Open the Anaconda prompt and enter the installation command to install SimpleITK for Anaconda. Open the Anaconda Navigator and launch Jupiter Notebook.
A web browser will open showing directories and files. Browse through the directory structures to reach the directory containing the downloaded Jupiter Notebook from GitHub. Then open the notebook and press Shift and Enter to run each cell.
In this representative experiment, adherent breast cancer cells were treated with an estrogen receptor agonist, or vehicle, for 24 hours. Spectrally separated probe sets against GREB1 intronic and exonic sequences allowed the simultaneous visualization and quantification of nascent and mature mRNA. Importantly, the number of transcriptionally active alleles in each cell that has been shown to be part of the estrogen response time course can be marked.
Counting the overlapping intron and exon spots, the E2-treated cells were determined to have a four-fold increased fraction of cells that exhibit two or more active GREB1 alleles compared to vehicle-treated cells. As introns are spliced out of nascent mRNA to produce mature mRNA that consists of only exon sequences, the number of mature mRNA per cell can be counted by quantifying the number of green spots. Plotting these data then allow the shift in distribution of mature GREB1 mRNA cell and the aggregate fold change in the cell population after E2 treatment to be observed.
C-FISH allows for investigating multiple RNA species sequentially, MERFISH is a barcoding technique to massively increase multiplexing, and iFISH is used to simultaneously study RNA and proteins.
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