A key area of interest in stem cell biology is understanding the mechanisms that regulate self renewal, differentiation, cell cycle control and other biological processes.

Post-transcriptional regulation by microRNA (miRNA) is one of several mechanisms under investigation. miRNA's are known to regulate the translation and stability of messenger RNAs (mRNA) by cleavage or by blocking the translation of mRNA into protein. Some miRNA's are differentially expressed in stem cells thereby providing unique molecular signatures by biological process, by cell type (embryonic, cancer or tissue-specific cells) and by species.

The Applied Biosystems miRNA Analysis Workflow is a complete system for miRNA profiling and functional analysis using Real-time PCR. The system offers several benefits over microarray-based solutions:

• Greater sensitivity to enable single cell analysis
• Simple, optimized workflow
• Scalable for global profiling and specific miRNA analysis

• Isolate miRNA: The TaqMan® MicroRNA Cells-to-CT™ Kit is a rapid lysis solution for cultured stem cells (10-1,000,000 cells/sample). It integrates cell lysis, DNA digestion and reverse transcription into a single tube and can be utilized with cells enriched by fluorescence-activated cell sorting (FACS). Alternatively, the mirVana™ miRNA Isolation Kit is designed to isolate total RNA from tissue and primary cells.
  
  
• Convert to cDNA: For cDNA synthesis from primary cells, use the TaqMan® MicroRNA Reverse Transcription Kit or Multiplex RT for TaqMan® MicroRNA Assays.
  
  
• Quantify miRNA Expression: Set up the individual reactions using the TaqMan® MicroRNA Assays or co-amplify more than 356 miRNA targets using the TaqMan® Array Human MicroRNA Panel. Both are fully optimized with the TaqMan® Universal PCR Master Mix and fully compatible with Applied Biosystems Real-Time PCR Instruments, including 7300, 7500, 7500 Fast, 7900HT Fast, StepOne™ and StepOnePlus™ Real-Time PCR Systems.
  
  
• Functional Analysis: Pre-designed Anti-miR™ miRNA Inhibitors and Pre-miR™ miRNA Precursors (miRNA mimics) can be introduced into cells to enable loss-of-function or gain-of-function studies to assess the biological impact of specific miRNAs. To analyze promoter activity in target genes, use the Reporter Gene Systems for in vivo or in vitro studies.
  
  

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