title: TaqMan Human MicroRNA Array v1.0 (Early Access)
status: Public on Jun 24 2010
contact.city: Toronto
contact.first: Patricia
contact.last: Reis
contact.email: preis@uhnres.utoronto.ca
contact.state: Ontario
contact.address: 610 University Avenue
contact.department: Applied Molecular Oncology
contact.country: Canada
contact.institute: University Health Network
organism: Homo sapiens
manufacture_protocol: Generation of miR expression data was a two-step process involving a reverse transcription reaction followed by a quantitative real-time polymerase chain reaction (qRT-PCR).  The reverse transcription reaction was carried out according to the manufacturer’s instructions including 1 mM dNTPs (including TTP), 50U MultiScribe Reverse Transcriptase, 3.8U RNase inhibitor, and 1x Reverse Transcription buffer.  The manufacturer suggests the use of 10-100ng/ul of input RNA for each reverse transcription reaction. TaqMan stem-loop primers for 365 individual miRs and 3 endogenous controls were used in the reverse transcription reaction.  Conditions for the reverse transcription reaction were as follows: 16˚C for 30 minutes, 42˚C for 30 minutes, 85˚C for 5 minutes, then hold at 4˚C.  cDNA samples were stored at -20˚C overnight prior to application on the TaqMan Human MicroRNA Array v1.0 (Early Access).  Reverse transcription reaction products were diluted (62.5x, 30x, 15x, or 5x) prior to adding an equivalent volume of TaqMan 2x Universal PCR Master Mix, No AmpErase UNG.  A minimum of 5x cDNA dilution is required to obtain the necessary volume for application to the TaqMan Human MicroRNA Array. The entire mixture was then added to individual ports of the TaqMan Human MicroRNA Array v1.0 (Early Access); the array was centrifuged at 1,200 rpm twice for 1 minute each, and then run on an Applied Biosystems 7900HT Fast Real-Time PCR system for 40 cycles.