A multisampling reporter system for monitoring microRNA activity in the same population of cells.
MicroRNAs (miRNAs) downregulate gene expression by binding to the partially complementary sites in the 3' untranslated region (UTR) of target mRNAs. Several methods, such as Northern blot analysis, quantitative real-time RT-PCR, microarray, and the luciferase reporter system, are commonly used to quantify the relative level or activity of miRNAs. The disadvantage of these methods is the requirement for cell lysis, which means that several sets of wells/dishes of cells must be prepared to monitor changes in miRNA activity in time-course studies. In this study, we developed a multisampling reporter system in which two secretable bioluminescence-generating enzymes are employed, one as a reporter and the other as an internal control. The reporters consist of a pair of vectors containing the Metridia luciferase gene, one with and one without a duplicated miRNA targeting sequence at their 3'UTR, while the other vector coding for the secreted alkaline phosphatase gene is used as an internal control. This method allows miRNA activity to be monitored within the same population of cells over time by withdrawing aliquots of the culture medium. The practicability and benefits of this system are addressed in this report.
Huang
Pei-Chen
PC
Institute of Biotechnology in Medicine, Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan.
Chen
Chih-Ying
CY
Yang
Feng-Yuan
FY
Au
Lo-Chun
LC
D002392:Catechin / Q000031:analogs & derivatives / Q000494:pharmacology; D017930:Genes, Reporter; D005821:Genetic Techniques; D056945:Hep G2 Cells; D006801:Humans; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism
C505362:MIRN16 microRNA, human; D035683:MicroRNAs; D002392:Catechin; C045651:epigallocatechin gallate