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, Cell nucleus, Transcription, RNA polymerase, Single molecule localization
Part I. Flow sorting of Arabidopsis interphase nuclei
Materials and Reagents
Equipment
Software
Procedure
Recipes
Part II. Immunolabeling of sorted nuclei
Part III. SIM and PALM evaluation
This protocol is based on using the ELYRA PS.1 system with the software ZEN 2012. Systems from other suppliers have similar functions. Therefore, the protocol can well serve also for other commercial systems including the processing steps. Being trained on such systems the resemblance of specific instructions to those given here should become obvious.
Notes
Acknowledgments
We thank Andrea Kunze and Joachim Bruder for excellent technical assistance. The protocol was developed based on previously published work of Schubert (2014), Schubert and Weisshart (2015).
References
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