Technology and Methodology
RNA Interference
RNA interference (RNAi) is a term typically used to encompass a set of epigenetic processes which inhibit the translation of protein from messenger RNA (mRNA) that has already been produced. The two main methods of inhibition are 1) destruction of the mRNA or 2) blockage mRNA so translation is stopped before the amino acid sequence is completed. RNAi is a post-translational gene regulation mechanism inherent to most cells.
Researchers 'hijack' the mechanism to target the proteins of interest. The method typically begins with the mRNA of interest. One determines a short (20-24 basepair) DNA sequence complementary to the target mRNA, but not complementary to other mRNA within the same genome. This sequence is used to form the 'stem' part of a 'stem-loop' structure, shaped sort of like a flat lollypop, called a short-hairpin RNA (shRNA). The loop is then cleaved off of the shRNA by an enzyme (e.g. Dicer), leaving the double-stranded stem containing the siRNA sequence. This stem is bound by a protein complex known as the RNA-Induced Silencing Complex (RISC). The RISC uses the siRNA sequence as its targeting 'key' to find the matching mRNA. Once that target mRNA is bound to the RISC, it is then suppressed using one of the methods described above. Which method is used depends on the design of the siRNA sequence.
Design of the siRNA sequence is often the most difficult part of the experiment. Suppression can vary widely from one sequence to another, although most researchers seem to find a sequence that provides at least 80% suppression when they test 4 sequences. One must also be aware of the likelihood of off-target effects even when the sequence may not be homologous with other genes of that species.
Rapid Prototyping of Microfluidic Devices
Microfluidic devices are fabricated using a micromolding technique. Channel networks are first designed using a drawing program (we use Adobe Illustrator) and output at 10,000 dpi to a transparency. The transparency can be used directly as a mask or the pattern can be transferred to a chrome mask for use in an aligner.
- First, a thick epoxy resin (epon) is deposited onto a silicon wafer. The mask is brought into intimate contact with the epon.
- After baking to facilitage cross-linking of the exposed areas, un-exposed epon is washed away with developer.
- With a patterned mold, poly(dimethyl siloxane) is mixed (10:1 elastomer:hardner) and potted onto the patterned substrate.
- The molded structure is then sealed to another layer of PDMS or glass after exosure to an oxygen plasma. Connection holes (arrow) are then bored through the PDMS.
- Connections may then be made to external fluid sources.
Fluid Control Systems
Taking advantage of the elastic character of the PDMS, it is possible to incorporate internal valves in a multi-layer microfluidic device. Specific geometry, combined with a pneumatically controlled valve system allows for high-speed actuation of valves and inlet channels. The controller is able to interface with a computer via a USB connection. Engineering software packages such as LabView and Matlab provide us with an efficient method of programming the device for different experimental studies.