Challenges:
mRNA is challenging to work with because it breaks down easily, it is autolytic, and there is an endogenous enzyme the is very robust that actively degrades RNA, RNAse. DNA also needs to be degraded so that these nucleic acids do not confound expression results. The mRNA must be extracted in a usable way for later analytical steps. Traditional methods use silica to selectively extract RNA and specialized inhibitors are added to arrest the activity of RNAse.
Current designs:
There are several options based on the lysing method: magnetic beads and chemical lysis. Don't worry picture will be coming soon. There are protocols I am looking into that do not require an isolation step, but we will be testing all protocols in a conventional lab setting before going micro.
mRNA is challenging to work with because it breaks down easily, it is autolytic, and there is an endogenous enzyme the is very robust that actively degrades RNA, RNAse. DNA also needs to be degraded so that these nucleic acids do not confound expression results. The mRNA must be extracted in a usable way for later analytical steps. Traditional methods use silica to selectively extract RNA and specialized inhibitors are added to arrest the activity of RNAse.
Current designs:
There are several options based on the lysing method: magnetic beads and chemical lysis. Don't worry picture will be coming soon. There are protocols I am looking into that do not require an isolation step, but we will be testing all protocols in a conventional lab setting before going micro.
- Magnetic Beads: Magnetic beads attract nucleic acids. When the spin of the magnetic beads speeds up to the point of lysis, the vortexing motion will cause cellular components to the outer edge of the device. Nucleic acids are bound to the beads when the solution is slightly acidic. The beads would take a path a shorter radius from the center because the attraction of the magnetic beads to the force of the spinning motor magnet ( like the magnet used in cooling systems for computers). Beads can be pulled through fluid that degrades DNA, and washes the nucleic acids of other contaminants, delivering the RNA to the final reaction well. RNA can be released from the beads in a slightly alkaline solution. As we mentioned in the previous module, magnetic beads can be difficult to work with. Secondly, a spinning component can cause a rotational spin in the final CanSat and that is another drawback. However, spin of the beads could be induced by a nonmoving electromagnet to avoids this issue.
- Chemical Lysis paired with Isotachophoresis: In isotachophoresis there is a finite injection of sample, between two phases of fluids. The leading electrolyte has the most ions of all three phases. The crude sample and the trailing electrolyte have similar analytes, but significantly fewer electrolytes, by design, than the leading electrolyte. These three phases are put under a current field of up to 500 V. Ideally the lysis chemical would keep the proteins in an uncharged oppositely charged state. Research has indicated that at lower pH's, the charge on proteins are neutral, while increasing the mobility of nucleic acids.
- Chemical Lysis paired with membraneless H-Filter: Mixing of fluids primarily occurs due to diffusion in microfluidic systems. Hence, two similar fluids can be channeled to flow next to each other, but the flows will only mix after a period of time, sometimes terribly long time. By selecting a fluid that attracts nucleic acids selectively to run adjacent to our cell lysate or cell culture medium and employing the diffusibility constant of RNA, we can calculate the path length the adjacent flows need to flow adjacently to allow for RNA to selectively diffuse. We do this using Pe'clet's dimensionless number equation. The bulk cell parts, the RNAse and media stay separate and flow into waste channels and the selective flow channel takes the RNA to the reaction wells. A dielectric charge can be applied to encourage the selective diffusion.