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Fluorescence in situ Localization of Pri-miRNAs in Isolated Arabidopsis thaliana Nuclei

拟南芥离体细胞核中 Pri-miRNA 的荧光原位定位

TG Tomasz Gulanicz
AZ Agnieszka Zienkiewicz
KZ Krzysztof Zienkiewicz
AK Anna Kasprowicz-Maluski
ZS Zofia Szweykowska-Kulinska
AJ Artur Jarmolowski

发布: 2023年09月20日第13卷第18期 DOI: 10.21769/BioProtoc.4824 浏览次数: 2278

评审: Durai SellegounderEduardo ListikShreya MukhopadhyayAnonymous reviewer(s)

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Abstract

Here, we present an approach combining fluorescence in situ hybridization (FISH) and immunolabeling for localization of pri-miRNAs in isolated nuclei of A. thaliana. The presented method utilizes specific DNA oligonucleotide probes, modified by addition of digoxigenin-labeled deoxynucleotides to its 3′ hydroxyl terminus by terminal deoxynucleotidyl transferase (TdT). The probes are then detected by immunolabeling of digoxigenin (DIG) using specific fluorescent-labeled antibodies to visualize hybridized probes. Recently, we have applied this method to localize pri-miRNA156a, pri-miRNA163, pri-miRNA393a, and pri-miRNA414 in the nuclei isolated from leaves of 4-week-old A. thaliana. The present approach can be easily implemented to analyze nuclear distribution of diverse RNA classes, including mRNAs and pri-miRNAs in isolated fixed cells or nuclei from plant.

Keywords: Fluorescent in situ hybridization (荧光原位杂交) search Immunolabeling (免疫标记) search Digoxigenin (地高辛) search Pri-miRNA (Pri-miRNA) search Confocal microscopy (共聚焦显微镜) search

Background

Microscopic imaging allows to observe and analyze the localization of diverse RNA molecules in their native cellular environment. It was found to be essential for understanding their turnover and function in different types of cells. Conventional fluorescence in situ hybridization (FISH) techniques have been designed for visualization of RNAs and relay on using fluorescently labeled DNA oligonucleotides (Hu et al., 2014). However, FISH methods are often challenging to detect RNAs expressed at low levels in the cells, such as pri-miRNAs, mainly due to low efficiency of resulting fluorescence signal. Here, we describe a method to detect pri-miRNA molecules in isolated nuclei of A. thaliana by combining FISH together with immunolocalization of digoxigenin (Figure 1). This method has been shown to be more sensitive and effective than conventional FISH (Bhat et al., 2020; Gonzalo et al., 2022; Stepien et al., 2022), and can successfully be applied to detect pri-miRNAs in plant and animal cells.



Figure 1. Overview of combined fluorescence in situ hybridization (FISH) and immunolabeling approach to detect pri-miRNAs in situ. Prior to the FISH, DNA oligonucleotides were labeled with digoxigenin at their 3′-ends by terminal transferase (TdT) (1), and the nuclei were isolated following the procedure described in (2). Pri-miRNAs were next localized by applying FISH (3) combined with the immunolocalization of digoxigenin attached to 3′-ends of the probes (4 and 5). The results were registered with confocal microscope (6). DIG: digoxigenin, TdT: terminal transferase, pAb: primary antibody, sAb: secondary antibody

Materials and reagents

Reagents

  1. Roche Terminal Transferase 8000 U (Merck, catalog number: 3333566001)

  2. ChromaTideTM Alexa FluorTM 488-5-dUTP (Thermo Fisher, catalog number: C11397)

  3. Roche DIUTPS-RO (DIG-11-dUTP) (Digoxigenin-11-dUTP, alkali-stable) (Merck, catalog number: 11093088910)

  4. Hoechst 33342, trihydrochloride, trihydrate, 100 mg (Thermo Fisher, catalog number: H1399)

  5. ProLongTM Gold Antifade Mountant (Thermo Fisher, catalog number: P36930)

  6. Paraformaldehyde, 16% w/v aq. soln., methanol free (Thermo Fisher, catalog number: 043368.9M)

  7. Formamide (Merck, catalog number: FX0420)

  8. Sodium chloride (Merck, catalog number: 1.16224)

  9. Potassium chloride (Merck, catalog number: 7447-40-7)

  10. Magnesium chloride (Merck, catalog number: 7786-30-3)

  11. Sodium citrate (Merck, catalog number: 1613859)

  12. Sodium phosphate, 0.5 M, pH 7.0 buffer (Thermo Fisher, catalog number: J63791.AK)

  13. Saline sodium citrate (SSC) 20× concentrate (Merck, catalog number: S6639)

  14. Ethylenediaminetetraacetic acid (EDTA) (Merck, catalog number: E9884-100G)

  15. Bovine serum albumin (BSA) (Merck, catalog number: A9418-5G)

  16. Ficoll® 400 (Merck, catalog number: F2637-5G)

  17. Polyvinylpyrrolidone (Merck, catalog number: P0930-50G)

  18. BSA, acetylated (Thermo Fisher, catalog number: AM2614)

  19. Spermine (Merck, catalog number: S3256-1G)

  20. Anti-digoxigenin antibody (Merck, 11333062910)

  21. Goat anti-mouse secondary antibody, Alexa Fluor Plus 488 (Thermo Fisher, catalog number: A55058)

  22. Goat anti-mouse secondary antibody, Alexa Fluor Plus 555 (Thermo Fisher, catalog number: A32727)

  23. RNase A (Thermo Fisher, catalog number: EN0531)

  24. Tris-HCl at pH 7.5 (Thermo Fisher, catalog number: 15567027)

  25. Tween 20 (Merck, catalog number: P1379)

  26. Sucrose (Merck, catalog number: S0389)

  27. Phosphate buffered saline (PBS) (Merck, catalog number: P4417)

  28. Triton X-100 (Merck, catalog number: 9036-19-5


Solutions

  1. Hybridization buffer (see Recipes)

  2. Denhardt’s solution 100× (50 mL) (see Recipes)

  3. LB01 buffer (10 mL) (see Recipes)

  4. Sorting buffer (10 mL) (see Recipes)

Equipment

  1. Humid chamber (e.g., StainTray Slide Staining, Polysciences, catalog number: 25502-1)

  2. Polylysine adhesion slides (e.g., Roth, catalog number: ET10.1)

  3. Cover glasses for microscope slides (e.g., Merck, Brand cover glasses, catalog number: BR470045)

  4. CellTrics 30 µm (Sysmex, catalog number: CTS220112)

  5. Confocal microscope, Leica DMI 6000B, TCS TrinoTCS SP8

  6. Plan Apochromat DIC H oil immersion lens (63×/1.4)

Software

  1. ImageJ, Fiji (https://imagej.net/software/fiji/)

Procedure

English
中文翻译

文章信息

版权信息

© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).

如何引用

Gulanicz, T., Zienkiewicz, A., Zienkiewicz, K., Kasprowicz-Maluski, A., Szweykowska-Kulinska, Z. and Jarmolowski, A. (2023). Fluorescence in situ Localization of Pri-miRNAs in Isolated Arabidopsis thaliana Nuclei. Bio-protocol 13(18): e4824. DOI: 10.21769/BioProtoc.4824.

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分类

分子生物学 > RNA > RNA 检测

植物科学 > 植物分子生物学 > RNA

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