Differential gene expression using mRNA isolated on plastic microfluidic chips.
Here we demonstrate the ability to perform differential gene expression experiments using messenger RNA (mRNA) isolated from crude cell lysates using a plastic microfluidic solid phase extraction column. The microfluidic columns (100microm by 100microm by 1.5 cm) were fabricated in a cyclic polyolefin by hot-embossing with an electroformed master-mold. The solid-phase consisted of a photopolymerized microporous monolith embedded with functional microparticles and covalently attached to the channel walls via photoinitiated grafting. For mRNA isolation from total RNA and direct mRNA isolation from cell lysates, oligo(dT) beads were embedded in the monolith. The extraction efficiency of the system is approximately 80% and the nucleic acid binding capacity of the silica solid-phase in this configuration is approximately 3.5 ng. The micro solid-phase was applied for the extraction and purification of mRNA from human liver total RNA and the isolation of mRNA from neonatal human dermal fibroblast cells (NHDF) and MCF7 breast cancer cell lysates. Differential gene expression between the two cell lines is demonstrated.
Bhattacharyya
Arpita
A
Department of Biomedical Engineering, Boston University, Boston, MA, USA. arpitabhattacharyya@gmail.com
Klapperich
Catherine M
CM
D004867:Equipment Design; D020869:Gene Expression Profiling / Q000295:instrumentation*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D010969:Plastics / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics*
D010969:Plastics; D012333:RNA, Messenger