Chapter 24
Transfection of Small Noncoding RNAs into Arabidopsis
thaliana Protoplasts
Ste ´ phanie Lalande, Marjorie Chery, Elodie Ubrig, Guillaume Hummel,
Julie Kubina, Ange ` le Geldreich, and Laurence Drouard
Abstract
Polyethylene glycol transfection of plant protoplasts represents an efficient method to incorporate foreign
DNA and study transient gene expression. Here, we describe an optimized protocol to deliver small
noncoding RNAs into Arabidopsis thaliana protoplasts. An example of application is provided by demonstrating the incorporation of a 20 nt long small noncoding RNA deriving from the 5
0 extremity of an
A. thaliana cytosolic alanine tRNA into freshly isolated protoplasts.
Key words Transfection, Small noncoding RNAs, tRNA-derived fragments, Protoplasts, Northern
blot
1 Introduction
Among the various classes of small noncoding RNAs (sncRNAs),
tRNA-derived fragments (tRFs) are now recognized as important
regulators of numerous biological processes [1–4]. In plants, tRFs
represent a large and dynamic repertoire of small RNA fragments
[5] but their molecular functions are still poorly understood
[2]. Among the different ways to decipher the molecular processes
in which tRFs are involved, the use of in vivo approaches is rather
limited. Indeed, the production of specific tRFs in transgenic plants
remains difficult to achieve. Facing this bottleneck, cell transfection
represents an interesting option. This technique, mainly developed
in the animal field to study the biology of miRNAs or siRNAs, is
based on the use of lipofectamine, a reagent that facilitates efficient
delivery of small RNA molecules into cells (see for instance [6, 7]).
In plants, to deliver nucleic acids, researchers have chosen to work
with plant protoplasts (i.e., cells without their cell wall). Nevertheless, chemicals such as lipofectamine appeared toxic and inefficient
[8] and thus are rarely utilized. Rather, three other types of
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_24, © Springer Science+Business Media, LLC, part of Springer Nature 2020
413
Transfection of Small Noncoding RNAs into Arabidopsis
thaliana Protoplasts
Ste ´ phanie Lalande, Marjorie Chery, Elodie Ubrig, Guillaume Hummel,
Julie Kubina, Ange ` le Geldreich, and Laurence Drouard
Abstract
Polyethylene glycol transfection of plant protoplasts represents an efficient method to incorporate foreign
DNA and study transient gene expression. Here, we describe an optimized protocol to deliver small
noncoding RNAs into Arabidopsis thaliana protoplasts. An example of application is provided by demonstrating the incorporation of a 20 nt long small noncoding RNA deriving from the 5
0 extremity of an
A. thaliana cytosolic alanine tRNA into freshly isolated protoplasts.
Key words Transfection, Small noncoding RNAs, tRNA-derived fragments, Protoplasts, Northern
blot
1 Introduction
Among the various classes of small noncoding RNAs (sncRNAs),
tRNA-derived fragments (tRFs) are now recognized as important
regulators of numerous biological processes [1–4]. In plants, tRFs
represent a large and dynamic repertoire of small RNA fragments
[5] but their molecular functions are still poorly understood
[2]. Among the different ways to decipher the molecular processes
in which tRFs are involved, the use of in vivo approaches is rather
limited. Indeed, the production of specific tRFs in transgenic plants
remains difficult to achieve. Facing this bottleneck, cell transfection
represents an interesting option. This technique, mainly developed
in the animal field to study the biology of miRNAs or siRNAs, is
based on the use of lipofectamine, a reagent that facilitates efficient
delivery of small RNA molecules into cells (see for instance [6, 7]).
In plants, to deliver nucleic acids, researchers have chosen to work
with plant protoplasts (i.e., cells without their cell wall). Nevertheless, chemicals such as lipofectamine appeared toxic and inefficient
[8] and thus are rarely utilized. Rather, three other types of
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_24, © Springer Science+Business Media, LLC, part of Springer Nature 2020
413