(111a) mRNA Extraction from Mycobacteria M. Smegmatis Utilizing Ultrahigh Field Intensity Electrolysis

The characterization of gene expression level has been the interest to researchers for a long time, both in specific development stage and physiological condition. Understanding the transcriptome, e.g. the complete set of transcripts in a cell, is essential for interpreting the functional elements of genome. Even though there is not necessarily always a strong correlation between the abundance of mRNA and related proteins, measuring mRNA levels is still informative in understanding how the transcriptional machinery of the cell is affected by external signal or how the cells interact with environment.

Real time PCR, microarrays and RNA sequencing (RNA-seq) have been developed in recent years to quantify the mRNA for understanding physiological events at the whole genome-level level. The purity and integrity of input RNA are critical for the success of these RNA based analysis. Any compromise in RNA quality can lead to variable results. However for some of the species, such as Mycobacteria, the evaluation of transcriptome has been hindered by the lysis procedures available. The thick lipid-rich cell wall and slow replication of mycobacteria make chemical disruption of the cells tedious and considerably difficult. Several methods have been reported for the isolation of RNA from mycobacteria, such as the Trizol-based method and the Hot-phenol based method. However, these methods use highly toxic chemicals in several steps and give variable quality of RNA. The typical lysis procedure which is achieved by lysozyme, proteinase K and sodium dodecyl sulfite (SDS) quantity of mRNA typically takes several days. The involved chemicals and physical force may result in the RNA fragmentation which would compromise the accuracy of mRNA evaluation. To minimize fragmentation, the extraction protocol should be rapid with minimal chemical involved. In addition, the efficiency of RNA extraction also needs to be improved to evaluate the absolute amount of RNA in individual cells. The conventional procedure is usually applied to a large number of cells (up to 10⁸ cells) which limits its application with limited amount of cells such as bacteria-host interaction or single cell analysis. Hence, it is important to develop new approach for mRNA extraction for mycobacteria, such as M. Smegmatis.

Electrical cell lysis shows great advantages of its fast speed and reagent procedure. The electrical lysis is based on electroporation which applies electrical pulses with defined voltages and widths causes the formation of nano-pores on the cell membrane. With increased voltages and duration of electrical pulses, the cells will be lysised and their intracellular materials will be released. To dealing with small number of cells, we conduct electrical lysis in the microfluidic chip. The major challenge of microfluidic electrical lysis is the generation of bubbles due to the water electrolysis. Here we describe a rapid mRNA extraction method utilizing ultra-high field intensity electrolysis. We are able to apply 6,000 V/cm dc field for rapid lysis from about 0.1 million M. Smegmatis cells. The mRNA extraction efficiency is about 6 times higher than state-of-the-art bead beating approach.

In our design, we designed a two layer PDMS microfluidic chip for M. Smegmatis electrical lysis. The depth of shallow part of the channel (2 μm) is designed to be smaller than microscale silica beads (4.8 μm in the diameter). The suspended microsphere will be stopped by the shallow channel and packed to a microscale matrix that stops the M. Smegmatis in the solution. The amount of packed beads is controlled by a pneumatic valve fabricate by multilayer soft lithography. The geometric modification was used to locally amplify the electrical field in the narrow section of the channel, while the rest of the channel is much wider so that the overall voltage required for lysis is relatively low.  Subsequent electrical pulses rapid lyse the cells and release intracellular proteins and mRNAs. The decrease of fluorescence from green fluorescent protein (GFP) released by GFP expressing M. Smegmatis is observed. The amount of released mRNA is quantified by quantitative reverse transcript polymerase chain reaction (qRT-PCR). In our approach, the capturing and lysis of cells are based on physical methods without involving chemical or biological reagent.

In our approach, the cell lysis procedure is extremely fast without using additional chemicals. The cell lysate can be directly used for qRT-PCR analysis without further treatment. Furthermore, our instrumentation is extremely simple. The electrical pulses was controlled by LabVIEW program and a relay combination. A high voltage power supply was used as a power source. The microfabrication process is simple and low cost. There is no microfabricated electrode used in our design. Finally, our approach can be easily scaled down to analysis small amount of cells and integrated with electrokinetics-based technology.