methods have been developed to transfect macromolecules: electroporation (e.g., [9]), microinjection (e.g., [10]), and polyethylene glycol (PEG)-mediated transfection (e.g., [11], the latter
method being the most widely used). Such protocols are often
employed to deliver DNA into protoplasts for transient gene
expression studies, but the incorporation of RNAs into plant protoplasts has also been developed. For instance, viral RNAs, siRNAs,
and tRNAs were electroporated into protoplasts [12, 13] and a
PEG method allowed viral RNA to be delivered [14]. Here, we
have adapted and optimized an efficient DNA transfection method
described in details by Yoo et al. [11] to incorporate sncRNAs into
Arabidopsis thaliana protoplasts. As an example of application, we
describe here the transfection of an A. thaliana 20 nt long tRF
alanine (tRF Ala20) [5] into protoplasts isolated either from an
A. thaliana cell culture or from young seedlings. To validate the
PEG transfection protocol, incorporation of tRF Ala20 was verified
by northern blot analysis of the small RNA population extracted
from RNase-treated transfected protoplasts. Therefore, we propose
this efficient RNA transfection method as a new tool to study the
biology of sncRNAs. For example, tagged (e.g., fluorophores, biotin) sncRNAs could be applied for in vivo live imaging or interactome studies.
2 Materials
2.1 Plant Material
1. Culture of individualized cells obtained from the Landsberg
A. thaliana ecotype.
2. A. thaliana Columbia (Col-0) ecotype seeds.
2.2 Nucleic Acids
1. 100 μM Synthetic oligoribonucleotide of interest dissolved in
diethyl pyrocarbonate (DEPC)-treated water at a 100 μM
concentration.
2. Any plasmid of small size (i.e., 3–4 kb).
3. 100 μM Oligodeoxynucleotide with a sequence complementary to the sequence of the small RNA of interest.
2.3 Solutions
All solutions should be prepared with RNase-free materials and
reagents, and with RNase-free, sterile, and ultrapure water. Solutions are autoclaved for 15 min at 120
C (except otherwise stated).
Procedures for preparing classical solutions can be found in Sambrook and Russell [15].
1. Cell culture medium: 4.41 g/L MS256, 30 g/L sucrose,
5.4 μM α-naphthaleneacetic acid (NAA), 0.23 μM kinetin,
pH 5.6. Weigh 4.41 g of MS256 and 30 g of sucrose. Transfer
to a glass beaker. Add about 500 mL of water. Mix well and
adjust pH to 5.6 with 1 N KOH. Add 100 μL of 10 mg/mL
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Ste ´ phanie Lalande et al.
method being the most widely used). Such protocols are often
employed to deliver DNA into protoplasts for transient gene
expression studies, but the incorporation of RNAs into plant protoplasts has also been developed. For instance, viral RNAs, siRNAs,
and tRNAs were electroporated into protoplasts [12, 13] and a
PEG method allowed viral RNA to be delivered [14]. Here, we
have adapted and optimized an efficient DNA transfection method
described in details by Yoo et al. [11] to incorporate sncRNAs into
Arabidopsis thaliana protoplasts. As an example of application, we
describe here the transfection of an A. thaliana 20 nt long tRF
alanine (tRF Ala20) [5] into protoplasts isolated either from an
A. thaliana cell culture or from young seedlings. To validate the
PEG transfection protocol, incorporation of tRF Ala20 was verified
by northern blot analysis of the small RNA population extracted
from RNase-treated transfected protoplasts. Therefore, we propose
this efficient RNA transfection method as a new tool to study the
biology of sncRNAs. For example, tagged (e.g., fluorophores, biotin) sncRNAs could be applied for in vivo live imaging or interactome studies.
2 Materials
2.1 Plant Material
1. Culture of individualized cells obtained from the Landsberg
A. thaliana ecotype.
2. A. thaliana Columbia (Col-0) ecotype seeds.
2.2 Nucleic Acids
1. 100 μM Synthetic oligoribonucleotide of interest dissolved in
diethyl pyrocarbonate (DEPC)-treated water at a 100 μM
concentration.
2. Any plasmid of small size (i.e., 3–4 kb).
3. 100 μM Oligodeoxynucleotide with a sequence complementary to the sequence of the small RNA of interest.
2.3 Solutions
All solutions should be prepared with RNase-free materials and
reagents, and with RNase-free, sterile, and ultrapure water. Solutions are autoclaved for 15 min at 120
C (except otherwise stated).
Procedures for preparing classical solutions can be found in Sambrook and Russell [15].
1. Cell culture medium: 4.41 g/L MS256, 30 g/L sucrose,
5.4 μM α-naphthaleneacetic acid (NAA), 0.23 μM kinetin,
pH 5.6. Weigh 4.41 g of MS256 and 30 g of sucrose. Transfer
to a glass beaker. Add about 500 mL of water. Mix well and
adjust pH to 5.6 with 1 N KOH. Add 100 μL of 10 mg/mL
414
Ste ´ phanie Lalande et al.