Analysis of mRNA populations from single live and fixed cells of the central nervous system.
This unit presents a method for the amplification of poly(A)(+) mRNA extracted from the cytoplasm of a single cell. After cDNA is synthesized from the mRNA, it is made double stranded, denatured, and reverse transcribed to yield antisense RNA (aRNA). Another round of amplification results in a relatively large amount of aRNAs in essentially the same proportion as in the starting mRNA population. RNA amplification protocols can be used for many purposes, including generation of disease expression profiles, making of cDNA libraries, and generation of diagnostics and therapeutics for disease. An alternate protocol is used to amplify RNAs from single neurons in fixed tissue specimens. Support protocols gives instructions for reverse northern analysis, which allows analysis of the presence or absence and relative levels of mRNA expression in selected cells, and a convenient method to assess the RNA content in fixed tissue sections using the fluorescent dye acridine orange (which binds single-stranded nucleic acids).
Eberwine
J
J
University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, USA.
Crino
P
P
D000818:Animals; D002478:Cells, Cultured; D002490:Central Nervous System / Q000737:chemistry / Q000166:cytology* / Q000502:physiology*; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D009924:Organ Culture Techniques; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*
D012333:RNA, Messenger