Abstract
Single-molecule RNA fluorescence in situ hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA (Raj et al., 2008; Lee et al., 2016a). We adapted this technique to visualize RNAs in the C. elegans whole adult worm or its germline, which enabled simultaneous recording of nascent transcripts at active transcription sites and mature mRNAs in the cytoplasm (Lee et al., 2013 and 2016b). Here we describe each step of the smFISH procedure, reagents, and microscope settings optimized for C. elegans extruded gonads.
Keywords: Active transcription site, mRNA, smFISH, sygl-1, C. elegans, Gonad
Background
smFISH enables direct and precise quantitation of mRNA in vivo. In addition, multiple RNA species can be scored simultaneously in the same cell by multiplexing smFISH probes. There have been previous publications using smFISH in C. elegans, but those studies used wide-field microscopy, which often has lower spatial resolution and requires additional image processing (e.g., image deconvolution) (Ji and van Oudenaarden, 2012). Here we describe an smFISH procedure optimized for the C. elegans germline tissue. Each step is detailed, including use of confocal microscopy to obtain precise measurements. Our protocol minimizes sample-to-sample variability and allows precise quantitation of mRNA and nascent transcripts.
Materials and Reagents
Equipment
Procedure
Note: Figure 1 shows a flowchart of the protocol. All spins are at 400 x g (2,000 rpm) for 30 sec unless otherwise stated and buffer recipes are listed at the end. Figure 1. Flowchart of the smFISH protocol for the C. elegans gonad
Data analysis
Representative smFISH image (Figure 3). Figure 3. sygl-1 smFISH at the distal gonad. The sygl-1 gene is a direct target of Notch transcriptional activation. SYGL-1 protein is a critical regulator for germline stem cell (GSC) maintenance. Individual nascent transcripts (bright white nuclear spots; overlay of yellow and magenta channel, indicated by arrowheads) and cytoplasmic mRNA (magenta) of sygl-1 are visualized with spectrally distinct smFISH probes. DAPI marks DNA (blue). Maximum Z-projection is shown. Dashed line marks gonadal outline. Scale bar = 5 µm. Modified from Lee et al. (2016b). Images taken using confocal microscopy do not require further image processing. However, images taken using wide-field (compound) microscopy must be deconvolved with carefully chosen image processing conditions (e.g., iterations). A caveat to the wide-field method is that over- or under-processed images can produce false-positive or false-negative signals. Using confocal images, the RNA spots can be systematically quantified with standard image processing (e.g., ImageJ) or published tools (e.g., MATLAB codes) (Mueller et al., 2013; Lee et al., 2016b). The wide-field microscope with a typical CCD camera (e.g., Hamamatsu C11440) generates microscopic images with the resolution high enough (pixel size: 0.1-0.2 µm) for identifying single RNA spots (Lee et al., 2016a). For confocal microscopy, we recommend using a pixel size similar to the size that works for wide-field microscopy (Abbaszadeh and Gavis, 2016; Lee et al., 2016b). As a first step of smFISH analysis, we quantify the size, shape (e.g., Gaussian distribution), signal intensities and subcellular location of detected RNA spots to validate singularity of detected RNA spots. In the Kimble lab, we consistently see nuclei containing one to four active transcription sites using intron-specific smFISH probes, with four matching the maximum number of loci of a gene (after DNA replication in S phase). The nascent RNAs are nuclear and colocalized to DAPI (or other DNA strain of choice). The mRNAs are cytoplasmic. Detected mRNA spots show tight signal distribution (C.V. < 0.5) in most RNA species, indicating that the detected spots are likely single RNA molecules (Lee et al., 2013 and 2016b). Control experiments such as detecting RNAs in the target gene knock-out background and using a different set of probes hybridizing the same target gene should be done to ensure the specificity of the probes to the target gene.
Notes
Recipes
Acknowledgments
This protocol has been adapted from Lee et al. (2016b). ESK was supported by the American Cancer Society–George F. Hamel Jr. Fellowship (PF-14-147-01-DDC). HSS was supported by an Ellison Medical Foundation Fellowship of the Life Science Research Foundation. TRL is supported by the National Science Foundation Graduate Research Fellowship Program (Grant No. DGE-1256259). JK is an Investigator of the Howard Hughes Medical Institute.
References
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