RNA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays.
The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high-quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microarray analysis and Illumina-Solexa sequencing (RNA-Seq) of inner ear organs, and for cloning of large transcripts, such as those for ion channels. Genetic sequences cloned with these procedures can be used for transient transfection of Xenopus kidney cell lines with fluorescent protein fusion constructs.
Biology Department, New Mexico State University, Las Cruces, NM, USA.
Casilda
C
Trujillo-Provencio
Biology Department, New Mexico State University, Las Cruces, NM, USA.
TuShun R
TR
Powers
Biology Department, New Mexico State University, Las Cruces, NM, USA.
David R
DR
Sultemeier
Biology Department, New Mexico State University, Las Cruces, NM, USA.
Daniel
D
Ramirez-Gordillo
Biology Department, New Mexico State University, Las Cruces, NM, USA. serrano@nmsu.edu.
Elba E
EE
Serrano
D000818:Animals; D003001:Cloning, Molecular; D007758:Ear, Inner / Q000737:chemistry*; D020869:Gene Expression Profiling / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D009928:Organ Specificity; D012313:RNA / Q000302:isolation & purification*; D017423:Sequence Analysis, RNA; D014722:Vestibule, Labyrinth / Q000737:chemistry; D014981:Xenopus / Q000235:genetics*
D012313:RNA
Auditory; Cloning; Heterologous gene expression; Microarray; RNA; RNA-Seq; Transcriptional profiling; Vestibular; Xenopus laevis; Xenopus tropicalis