Micro RNA profiling: an easy and rapid method to screen and characterize stem cell populations.
MicroRNAs (miRNAs) are small regulatory RNAs varying in length between 20 and 24 nucleotides. They are thought to play a key role during development by negative gene regulation at the post-transcriptional level. Recent studies using quantitative polymerase chain reaction (QPCR) and northern blot analysis have reported the presence of several miRNA unique to specific cell types. The NCode multispecies miRNA array provides a means for simultaneously profiling the expression patterns of hundreds of known miRNAs in a given cell type or biological sample. Using this method, miRNA expression patterns in embryonic and adult stem cell lines can be characterized and compared with each other. The accuracy of NCode miRNA array data can be further confirmed by QPCR analysis of putative array hits. This array-based screening platform is a fast and easy to use analytical tool that allows one to asses the state of stem cell lines following multiple passages in culture as well as a discovery tool that eliminates the need to screen large numbers of candidate regulatory miRNAs by northern blot or PCR. In this chapter, we describe in detail the method to carry out miRNA array analysis in human embryonal carcinoma cells and confirm the array results using QPCR.
Lakshmipathy
Uma
U
Stem Cell Institute, University of Minnesota, Minneapolis, USA.
Love
Brad
B
Adams
Christopher
C
Thyagarajan
Bhaskar
B
Chesnut
Jonathan D
JD
D015152:Blotting, Northern / Q000379:methods*; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D035683:MicroRNAs; D012333:RNA, Messenger