In vivo modulation and quantification of microRNAs during axolotl tail regeneration.
The ability to regenerate diseased, injured, or missing complex tissue is widespread throughout lower vertebrates and invertebrates; however, our knowledge of the molecular mechanisms that regulate this amazing ability is still in its infancy. Many recent papers have shown important roles for microRNAs in regulating regeneration in a number of species. The ability to detect and quantify miRNA expression fluctuations at a single cell level in vivo in different cell types during processes like regeneration is very informative. In this chapter, we describe how to use a dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmid to quantitate the dynamics of specific miRNAs over time following miRNA mimic injection as well as during regeneration. In this bicistronic vector, the mRFP allows for verification of miRNA expression, while the GFP functions as an internal control to normalize miRNA expression and thus obtain quantitative results. In addition, we demonstrate how this technique revealed dynamic miR-23a expression and function during tail regeneration.
Department of Genetics, Cell Biology and Development, Stem Cell Institute, University of Minnesota, 6-160 Jackson hall, 321 Church St. SE, Minneapolis, MN, 55455, USA.
Jami R
JR
Erickson
Karen
K
Echeverri
D000558:Ambystoma mexicanum / Q000235:genetics* / Q000502:physiology*; D000818:Animals; D018274:Electroporation; D020869:Gene Expression Profiling / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007267:Injections; D008164:Luminescent Proteins / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D012038:Regeneration / Q000235:genetics*; D013623:Tail / Q000502:physiology*; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D035683:MicroRNAs; D000097573:Red Fluorescent Protein