mRNA isolation in a microfluidic device for eventual integration of cDNA library construction.
mRNA isolation for the purpose of cDNA library construction was performed in a microfluidic chip device using paramagnetic oligo-dT beads. The simple Y-intersection flow design mixes beads and sample on-chip, and uses magnetic trapping to capture, then release the beads. The capillary gel electrophoresis (CGE) detection of the total unamplified mRNA isolated on-chip, and of a reverse transcription-polymerase chain reaction (RT-PCR) amplified rare gene indicated that mRNA could be captured by oligo-dT beads on-chip, had very good integrity and was suitable for constructing a cDNA library. The limit of detection for the rare bicoid gene of Drosophila Melanogaster corresponded to the capture of approximately 2.8 ng of total mRNA from 0.85 microgram of total RNA (TRNA) within the microchip. As much as 34 ng of total mRNA was estimated to be captured from 10 micrograms of TRNA.
Jiang
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Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Harrison
D J
DJ
D000818:Animals; D004331:Drosophila melanogaster / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods; D015723:Gene Library; D017344:Genes, Insect; D008280:Magnetics; D008863:Microspheres; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger