Cloning of stress-responsive microRNAs and other small RNAs from plants.
In plants, small RNA (microRNAs and other endogenous small RNAs)-guided target gene expression is vital for a wide variety of biological processes including adaptation to stress conditions. Identification of stress-regulated microRNAs or other classes of endogenous small RNAs advances our understanding of post-transcriptional gene regulation important for plant stress tolerance. This chapter describes a detailed step-by-step protocol for cloning of small RNAs. Following 5' and 3' adapter ligation to the purified small RNAs, cDNA will be synthesized using reverse transcription, which will be further amplified using a polymerase chain reaction. The resulting small DNA fragments can be either cloned and sequenced using traditional sequencing method or subjected to direct high-throughput pyrosequencing or sequencing-by-synthesis technology.
Reyes
Jose Luis
JL
Department Biologia Molecular de Plantas, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Cuernavaca, Morelos, Mexico.
Arenas-Huertero
Catalina
C
Sunkar
Ramanjulu
R
D003001:Cloning, Molecular / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D018506:Gene Expression Regulation, Plant; D035683:MicroRNAs / Q000235:genetics* / Q000302:isolation & purification; D009691:Nucleic Acid Denaturation / Q000235:genetics; D027805:Phaseolus / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013312:Stress, Physiological / Q000235:genetics*
D035683:MicroRNAs; D018749:RNA, Plant