使用结构照明显微镜（SIM）和光敏定位显微镜(PALM）分析RNA聚合酶II分子在流式分选的拟南芥细胞核中的丰度及分布

Klaus Weisshart; Jörg Fuchs; Veit Schubert

doi:10.21769/BioProtoc.1725

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Structured Illumination Microscopy (SIM) and Photoactivated Localization Microscopy (PALM) to Analyze the Abundance and Distribution of RNA Polymerase II Molecules on Flow-sorted Arabidopsis Nuclei

使用结构照明显微镜（SIM）和光敏定位显微镜(PALM）分析RNA聚合酶II分子在流式分选的拟南芥细胞核中的丰度及分布

Klaus Weisshart

JF Jörg Fuchs

VS Veit Schubert email

发布: 2016年02月05日第6卷第3期 DOI: 10.21769/BioProtoc.1725 浏览次数: 12788

评审: Samik BhattacharyaAnonymous reviewer(s)

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Cover of Journal of Experimental Botany, featuring study using the protocol.

Mar 2015

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Abstract

RNA polymerase II (RNAPII) is the enzyme transcribing most of the eukaryotic protein-coding genes. Analysing the distribution and quantification of RNAPII can help understanding its function in interphase nuclei. Although several investigations in mammals indicate the organization of RNAPII in so-called ‘transcription factories’ (Jackson et al., 1993; Rieder et al., 2012; Papantonis and Cook, 2013), their existence is still controversially discussed (Zhao et al., 2014).

Recently, based on super-resolution microscopy the presence of transcription factories was also suggested in plants. Applying structured illumination microscopy (SIM) and photoactivated localization microscopy (PALM) the distribution and number of RNAPII molecules in Arabidopsis nuclei were analysed and a positive correlation between RNAPII abundance and endopolyploidy was found (Schubert, 2014; Schubert and Weisshart, 2015).

Here, we present a protocol describing the isolation of Arabidopsis thaliana interphase nuclei via flow-sorting according to their endopolyploidy level, followed by a double immunostaining using antibodies specific for different RNAPII modifications and the subsequent evaluation by spatial SIM and PALM to achieve results regarding the abundance, distribution and co-localization of single inactive and active RNAPII molecules.

Keywords: Super-resolution microscopy (超高分辨率显微镜)

Cell nucleus (细胞核)

Transcription (转录)

RNA polymerase (RNA聚合酶)

Single molecule localization (单分子定位)

Part I. Flow sorting of Arabidopsis interphase nuclei

Materials and Reagents

22x22 mm high precision coverslips (170±5 µm, no. 1.5H) (Marienfeld-Superior) Rubber cement (Marabu Australia, catalog number: 290110000 )
5 ml Polystyrene Round-Bottom Tubes with 35 µm cell strainer cap (BD Biosciences, catalog number: 352235 ) or alternatively, 5 ml Polystyrene Round-Bottom Tube (BD Biosciences, catalog number: 352063 ) in combination with disposable filters CellTrics, 30 or 50 µm (Sysmex-Partec, catalog numbers: 04-0042-2316 or 04-0042-2317 )
Note: Currently, it is "Corning, Falcon^®, catalog number: 352235” and "Corning, Falcon^®, catalog number: 352063”.
0.22 µm filter
A. thaliana rosette leaves
Calibration beads [Sphero^TM Rainbow Calibration Particles (8 peaks)] (BD Biosciences, catalog number: 559123 )
Accudrop Beads (BD FACS^TM Accudrop Beads) (BD Biosciences, catalog number: 345249 )
Formaldehyde solution 37% (Carl Roth GmbH + Co, catalog number: 7398.1 )
4', 6-diamidino-2-phenylindole (DAPI) (Life Technologies, Molecular Probes^TM, catalog number: D-1306 )
Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes^TM, catalog number: D-1306”.
Milli-Q water
NaCl
Na₂HPO₄^.2H₂O
NaH₂PO₄
Tris
Na₂EDTA
Spermin
KCl
Triton X-100
NaOH
β-mercaptoethanol
MgCl₂
Sucrose
Tween 20
HCl
BD FACSFlow sheath fluid (BD Biosciences, catalog number: 342003 ) or alternatively, 1 x PBS (see Recipes)
Formaldehyde fixative (see Recipes)
500 ml Tris-Buffer (see Recipes)
200 ml lysis buffer LB01 (Dolezel et al., 1989) (see Recipes)
100 µg/ml DAPI stock solution (see Recipes)
25 ml sucrose buffer (see Recipes)

Equipment

FACSAria IIu (BD Biosciences)

Software

FACSDiva software

Procedure

English

中文翻译

文章信息

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如何引用

Weisshart, K., Fuchs, J. and Schubert, V. (2016). Structured Illumination Microscopy (SIM) and Photoactivated Localization Microscopy (PALM) to Analyze the Abundance and Distribution of RNA Polymerase II Molecules on Flow-sorted Arabidopsis Nuclei. Bio-protocol 6(3): e1725. DOI: 10.21769/BioProtoc.1725.