220-plex microRNA expression profile of a single cell.
Here we describe a protocol for the detection of the microRNA (miRNA) expression profile of a single cell by stem-looped real-time PCR, which is specific to mature miRNAs. A single cell is first lysed by heat treatment without further purification. Then, 220 known miRNAs are reverse transcribed into corresponding cDNAs by stem-looped primers. This is followed by an initial PCR step to amplify the cDNAs and generate enough material to permit separate multiplex detection. The diluted initial PCR product is used as a template to check individual miRNA expression by real-time PCR. This sensitive technique permits miRNA expression profiling from a single cell, and allows analysis of a few cells from early embryos as well as individual cells (such as stem cells). It can also be used when only nanogram amounts of rare samples are available. The protocol can be completed in 7 d.
Tang
Fuchou
F
Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK.
Hajkova
Petra
P
Barton
Sheila C
SC
O'Carroll
Dónal
D
Lee
Caroline
C
Lao
Kaiqin
K
Surani
M Azim
MA
D000818:Animals; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription / Q000235:genetics
D017931:DNA Primers; D035683:MicroRNAs