Purification processes that are considered conventional are often labor-intensive. The costs of the operations are also very high because of the larger-scale automated purification. Generally, a requirement for performing downstream analysis is the purified nucleic acids, including quantitative PCR, DNA/RNA sequencing, and reverse transcriptase quantitative PCR. the process to eliminate DNA/RNA purification typically uses lysis master mixtures that prevent nucleic acid’s degradation. The biology products in those mixtures don’t have PCR/RT-PCR. However, some cells contain more robust cell membranes, making the lysis buffer incompatible with downstream PCR. (Xu Shi et al. #)
Magnetic beads can be used to purify DNA/RNA to retain and elute nucleic acids. This can be compatible and work parallelly with microfluidic platforms. The magnetic beads are available commercially with a variety of options. Beads connected with nucleic acids can be immobilized easily by a magnet, eliminating the need for fabricating structures for on-chip columns. Recently, the combination of magnetic beads and microdroplets is gaining the interest of researchers. The beads penetrate the droplet interface with the use of an external magnetic field, leading to a class of devices that doesn’t need extensive microchannel ways for manipulation of the liquid. An alternative way to use a droplet-based method is to specially design structures capable of holding the droplet but lets the magnetic beads travel through the gaps in it.
Following is a demonstration of how this approach efficiently purifies RNA. This process has a higher RNA recovery efficiency and results in single-cell level sensitivity.
Materials And Methods
Setup
The key components include a TREDA chip, a movable Plexiglas plate containing linear arrays of 16 extraction magnets below TREDA, and another one of Plexiglas plates with eight mixing magnets above TREDA. TEDRA chip is designed to hold 16 droplets where each set compromises of 3 droplets . one of them is used as a mixture for lysis, the second one as a wash buffer, and the last one for elution buffer. All three are submerged in an open pool of mineral oil, resulting in a unique formation.
Magnetic-Bead Extraction
The switchable magnetic beads carry positive charges if the pH around them is lower than 6.0 and negative charges when the pH rises up to 8.0. thi s causes the beads to be either bound or released from the surface depending on the charge. This is where the buffers are used. The lysis buffer, binding buffer, and beads are combined for cell lysis and RNA binding. Next, the washing buffer is used to wash out any unwanted substances from the beads. The beads are then removed from a low-salt, high pH elution buffer, and we are left with purified RNA in the elution buffer.
Conventional RNA Extraction Procedure
A freshly prepared lysis mix that includes precise amounts of lysis buffer and other molecular bioproducts is used in the conventional approach. We submerge the beads, are added to cell lysis and mixed using a pipette. A tight pellet of beads is formed, which is then washed with a wash buffer. The final elution is achieved by adding a precise amount of elution buffer.
TREDA Chip RNA Extraction Procedure
Sixteen sets of each droplet are pipetted onto the TREDA chip. The first droplet in each set contained a lysis mix, binding buffer, cell medium, and suspension of magnetic beads. The second and third droplets in the same set, respectively, contained washing buffer and elution buffer. After the drops were successfully pipetted on the TREDA chip, they were submerged in mineral oil in order to fully cover the droplets inside. The oil prevents the droplets from contamination or evaporation. To further validate the RNA extraction efficiency, the liquid from the third droplet was carefully placed in a PCR tube.
The Conclusion
The blog took you through the process of magnetic bead and droplet array as a way of testing RNA purification. We see consistent, sensitive data when the RT-qPCR reactions are performed on purified RNA. the result have low variation, which confirms the high quality of RNA when purified on the TREDA chip. The simplicity of both constructional and operational aspects of the chip makes for an excellent tool for the purification of RNA. It makes the purification efficient with the significant time saving and reduces human handled steps resulting in low human errors.
The droplet-based purification method has application in various fields, such as in measuring quantitative gene expression. The ideal design makes it easy to pair with qPCR arrays that make use of surface adhering droplets. Additionally, this device is able to provide high-quality results of purifies RNA for many other analyses.
Bibliography: Xu Shi, et al. Parallel RNA extraction using magnetic beads and a droplet array. Royal Society Of Chemistry, 18th December 2014, Retrieved August 30, 2021, from
https://pubs.rsc.org/en/content/articlehtml/2015/lc/c4lc01111b
