Biology Reference
In-Depth Information
CHAPTER
5
Regulation of Tubulin
Expression by Micro-RNAs:
Implications for Drug
Resistance
Sharon Lobert and Mary E. Graichen
School of Nursing, University of Mississippi Medical Center, Jackson, Mississippi, USA
CHAPTER OUTLINE
Introduction and Rationale......................................................................................... 64
5.1 Methods and Materials....................................................................................... 65
5.1.1 Measuring micro-RNAs ..................................................................... 65
5.1.1.1 Choice of Cell Line......................................................................65
5.1.1.2 RNA Extraction and qRT-PCR.....................................................65
5.1.1.3 PCR Arrays.................................................................................66
5.1.1.4 Next-Generation Sequencing.......................................................66
5.1.2 Upregulating micro-RNAs: Transfections with Precursor PremiRNAs ..... 67
5.1.3 Measuring Activity of Micro-RNAs...................................................... 67
5.1.3.1 Measuring micro-RNA Targets ....................................................67
5.1.3.2 Immunoprecipitation of RISC ......................................................67
5.2 Results and Discussion ...................................................................................... 68
5.2.1 b-Tubulin Isotypes and miR-100 ....................................................... 68
5.2.2 b-Tubulin Isotypes and miR-200c...................................................... 68
Summary .................................................................................................................. 70
References ............................................................................................................... 73
Abstract
In this chapter, we provide an overview of methods for studying micro-RNA regu-
lation of tubulin isotypes. In clinical studies,
b
-tubulin isotypes were found to be bio-
markers for tumor formation. In addition, because changes in the levels of specific
b
-tubulin isotypes alter the stability of microtubules in mitotic spindles
in vitro
, it has
been hypothesized that changes in microtubule protein levels could contribute to
chemotherapy resistance. Over the past 15 years, micro-RNAs have been shown
