16. To increase signal with X-ray exposure, use a reflective screen
placed on the film and incubate the cassette at À80
C. Under
these conditions, a strong signal can be obtained after 6 h of
exposure. At room temperature, an exposure time of 24–48 h is
recommended.
References
1. Shen Y, Yu X, Zhu L, Li T, Yan Z, Guo J
(2018) Transfer RNA-derived fragments and
tRNA halves: biogenesis, biological functions
and their roles in diseases. J Mol Med
96:1167–1176
2. Megel C, Morelle G, Lalande S, Duche ˆne A-M,
Small I, Mare ´chal-Drouard L (2015) Surveillance and cleavage of eukaryotic tRNAs. Int J
Mol Sci 16:1873–1893
3. Schimmel P (2018) RNA processing and modifications: the emerging complexity of the
tRNA world: mammalian tRNAs beyond protein synthesis. Nat Rev Mol Cell Biol 19:45–58
4. Cristodero M, Polacek N (2017) The multifaceted regulatory potential of tRNA-derived
fragments. Noncoding RNA Investig 1:7
5. Cognat V, Morelle G, Megel C, Lalande S,
Molinier J, Vincent T, Small I, Duche ˆne A-M,
Mare ´chal-Drouard L (2017) The nuclear and
organellar tRNA-derived RNA fragment population in Arabidopsis thaliana is highly
dynamic. Nucleic Acids Res 45:3460–3472
6. Haas G, Cetin S, Messmer M, Chane-WoonMing B, Terenzi O, Chicher J, Kuhn L,
Hammann P, Pfeffer S (2016) Identification
of factors involved in target RNA-directed
microRNA degradation. Nucleic Acids Res
44:2873–2887
7. Han Z, Ge X, Tan J, Chen F, Gao H, Lei P,
Zhang J (2015) Establishment of lipofection
protocol for efficient miR-21 transfection into
cortical neurons in vitro. DNA Cell Biol
34:703–709
8. Spo ¨rlein B, Koop H-U (1991) Lipofectin:
direct gene transfer to higher plants using cationic liposomes. Theor Appl Genet 83:1–5
9. Miao Y, Jiang L (2007) Transient expression of
fluorescent fusion proteins in protoplasts of
suspension cultured cells. Nat Protoc
2:2348–2353
10. Masani MYA, Noll GA, Parveez GKA,
Sambanthamurthi R, Pru ¨fer D (2014) Efficient
transformation of oil palm protoplasts by
PEG-mediated transfection and DNA microinjection. PLoS One 9:e96831
11. Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system
for transient gene expression analysis. Nat Protoc 2:1565–1572
12. Qi Y, Zhong X, Itaya A, Ding B (2004) Dissecting RNA silencing in protoplasts uncovers
novel effects of viral suppressors on the silencing pathway at the cellular level. Nucleic Acids
Res 32:e179
13. Wintz H, Dietrich A (1996) Electroporation of
small RNAs into plant protoplasts: mitochondrial uptake of transfer RNAs. Biochem Biophys Res Commun 223:204–210
14. McCormack JC, Simon AE (2006) Callus cultures of Arabidopsis. Curr Protoc Microbiol
00:16D.1.1–16D.1.9
15. Sambrook J, Russell DW (2001) In: Harbor
CS (ed) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY
Plant Protoplasts-Based System to Transfect RNA
429
placed on the film and incubate the cassette at À80
C. Under
these conditions, a strong signal can be obtained after 6 h of
exposure. At room temperature, an exposure time of 24–48 h is
recommended.
References
1. Shen Y, Yu X, Zhu L, Li T, Yan Z, Guo J
(2018) Transfer RNA-derived fragments and
tRNA halves: biogenesis, biological functions
and their roles in diseases. J Mol Med
96:1167–1176
2. Megel C, Morelle G, Lalande S, Duche ˆne A-M,
Small I, Mare ´chal-Drouard L (2015) Surveillance and cleavage of eukaryotic tRNAs. Int J
Mol Sci 16:1873–1893
3. Schimmel P (2018) RNA processing and modifications: the emerging complexity of the
tRNA world: mammalian tRNAs beyond protein synthesis. Nat Rev Mol Cell Biol 19:45–58
4. Cristodero M, Polacek N (2017) The multifaceted regulatory potential of tRNA-derived
fragments. Noncoding RNA Investig 1:7
5. Cognat V, Morelle G, Megel C, Lalande S,
Molinier J, Vincent T, Small I, Duche ˆne A-M,
Mare ´chal-Drouard L (2017) The nuclear and
organellar tRNA-derived RNA fragment population in Arabidopsis thaliana is highly
dynamic. Nucleic Acids Res 45:3460–3472
6. Haas G, Cetin S, Messmer M, Chane-WoonMing B, Terenzi O, Chicher J, Kuhn L,
Hammann P, Pfeffer S (2016) Identification
of factors involved in target RNA-directed
microRNA degradation. Nucleic Acids Res
44:2873–2887
7. Han Z, Ge X, Tan J, Chen F, Gao H, Lei P,
Zhang J (2015) Establishment of lipofection
protocol for efficient miR-21 transfection into
cortical neurons in vitro. DNA Cell Biol
34:703–709
8. Spo ¨rlein B, Koop H-U (1991) Lipofectin:
direct gene transfer to higher plants using cationic liposomes. Theor Appl Genet 83:1–5
9. Miao Y, Jiang L (2007) Transient expression of
fluorescent fusion proteins in protoplasts of
suspension cultured cells. Nat Protoc
2:2348–2353
10. Masani MYA, Noll GA, Parveez GKA,
Sambanthamurthi R, Pru ¨fer D (2014) Efficient
transformation of oil palm protoplasts by
PEG-mediated transfection and DNA microinjection. PLoS One 9:e96831
11. Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system
for transient gene expression analysis. Nat Protoc 2:1565–1572
12. Qi Y, Zhong X, Itaya A, Ding B (2004) Dissecting RNA silencing in protoplasts uncovers
novel effects of viral suppressors on the silencing pathway at the cellular level. Nucleic Acids
Res 32:e179
13. Wintz H, Dietrich A (1996) Electroporation of
small RNAs into plant protoplasts: mitochondrial uptake of transfer RNAs. Biochem Biophys Res Commun 223:204–210
14. McCormack JC, Simon AE (2006) Callus cultures of Arabidopsis. Curr Protoc Microbiol
00:16D.1.1–16D.1.9
15. Sambrook J, Russell DW (2001) In: Harbor
CS (ed) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY
Plant Protoplasts-Based System to Transfect RNA
429