sRNA-FISH: versatile fluorescent in situ detection of small RNAs in plants

Abstract

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA-FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co-localization of miR2275 and a 24-nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi-photon fluorescence excitation microscopy can be used to separate the target sRNA-FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA-FISH signals can be imaged using super-resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super-resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step-by-step sRNA-FISH protocol for studying sRNAs at the cellular and even subcellular level.

Keywords: Litchi chinensis; Oryza sativa; Zea mays; sRNA; LNA probes; fluorescent in situ hybridization; immunofluorescence; microRNA; multi-photon microscopy; technical advance.

Figure 1.. Workflow of sRNA-FISH.

Starting with sample preparation and probe design, tissues were fixed, embedded, sectioned and adhered to glass slides. Critical steps include determining sample autofluorescence and choosing antibodies with the right fluorophore combinations. After imaging, linear spectral unmixing is necessary for precise localization of sRNAs.

Figure 2.. Comparison of sRNA-FISH and traditional non-fluorescent in situ hybridization.

Probes reverse complement with zma-miR2275, zma-miR2118 and scrambled control were used to hybridize to maize anthers (~1 mm and ~0.4 mm in length respectively). The fluorescent micrographs were taken with laser scanning confocal microscopy. miR2275 localized mostly in tapetal and archesporial cells (top row, red), and miR2118 localized to cells in the epidermal layer (middle row, red). No signal was detected in the scramble control (bottom row). The background (blue) was spectrally unmixed from AF568 fluorescence. The micrographs of the non-fluorescent, colorimetric in situs were taken with bright-field microscopy and have the same localization pattern. EPI, epidermis; EN, endothecium; ML, middle layer; TA, tapetal layer; AR, archesporial cells. Scale bars = 20 μm for all images.

Figure 3.. Dual-target sRNA-FISH for maize anthers.

(a) sRNA-FISH detected both zma-miR2275 (detected in the AF633 channel; magenta) and the 24 nt phasiRNA (detected in the AF568 channel; cyan) in the tapetal layer and archesporial cells. Each image was collected in spectra mode with laser scanning confocal microscopy and then spectrally unmixed using Zen Software. Bright-field and merged images were also shown for each image. TA, tapetal layer; AR, archesporial cells. Scale bars = 20 μm for all images. (b) Quantification of the AF633 and AF568 signal intensity in dual-target sRNA FISH and controls. (Significance level: < 0.05, *; < 0.01, **).

Figure 4.. Comparison of one-photon and multi-photon excitation in litchi anthers.

(a) Images show 24 nt-phasiRNA localization in litchi, stage IV anthers. In images acquired with one-photon excitation, the spectra from strong autofluorescence (red) overlaps with AF647 signal. In images acquired with multi-photon excitation, distinct AF568 can be spectrally unmixed from background autofluorescence (both red and blue). TA, tapetal layer; AR, archesporial cells. Scale bar = 20 μm for all images. (b) Spectra profile of AF568/AF647 and litchi anther autofluorescence using one-photon and multi-photon excitation. Both AF568 and AF647 spectra are very close to background autofluorescence using one-photon excitation. AF568 has a distinct spectra compared with background autofluorescence using multi-photon excitation.

Figure 5.. Localization of miR2275 in premeiotic maize anthers using SIM.

Top left panel: Laser wide-field images shown miR2275 is detected in the archesporial cells and secondary parietal cells; the latter give rise to the middle layer and tapetum. Bottom left panel: detection of miR2275 using super-resolution structured illumination. miR2275 is localized to archesporial and secondary parietal cells. Right panels are images of the scrambled probe control. AR, archesporial cells; SPC, secondary parietal cells; EN, endothecium; EPI, epidermis. Scale bar = 20 μm for all images.

Figure 6.. Localization of miR2275 in premeiotic maize anthers using dSTORM.

(a) miR2275 was detected in the tapetal layer and archesporial cells. In comparison, scrambled probe yield very low signal. (b) Higher magnification images of box #1 and 2 showing localization around nucleus (Nu), and in the cytosol (Cy). TA, tapetal layer; AR, archesporial cells.