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More effective extraction of small RNAs

Small RNAs are gaining in importance in research as well as in the biotechnology and pharmaceutical industries. However, the potential of these molecules can only be exploited fully if very pure RNAs can be extracted from the cells in sufficiently high quantities. Currently used methods are expensive and only designed for large cell quantities. In addition, the operation of the extraction systems is very complicated. A new biochip, developed by Dr. Paul Vulto at the Institute of Microsystems Engineering in Freiburg, is able to extract RNAs from cells in a radically different way. His method is already being used by a pharmaceutical company.

RNAs are important cell constituents that make sure that genetic information is copied from the DNA and translated into proteins. However, there is increasing evidence that small RNAs also regulate the activity of many genes. This might be of great interest for those studying cancer cells. If researchers and pharmaceutical companies manage to manipulate the functions of these molecules, then they might, at some time in the future, be able to halt tumour growth. However, the short nucleic acids are quite difficult to handle. They must be isolated from the cells and purified to test their potential. And this is difficult with currently available methods. “Standard laboratory kits involve many individual steps to lyse the cells and subsequently separate the cellular content,” said Dr. Paul Vulto who has just finished his doctoral thesis in the department of Prof. Dr. Gerald Urban at the Freiburg Institute of Microsystems Engineering (IMTEK). “The extraction and purification of RNA typically takes up to four hours.”

Only minutes from the goal

RNA extraction on a fingertip: this biochip enables the automated separation of small RNAs from other cell constituents. © Dr. Paul Vulto

Apart from the complicated operation and the loss of time, the individual work steps cannot easily be automated. This would require expensive robots. However, automation is essential because pharmaceutical companies must be able to test thousands of substances in a very short time. Vulto and his colleagues at the IMTEK have now solved these problems in an ingenious way. Vulto studied electrical engineering in the Netherlands and developed a biochip during his doctorate that was no larger than a fingertip. The procedure, which previously took four hours, now only requires about ten minutes. At first sight, the chip does not look very complicated. The use of the chip is also very easy: cancer cells are put into a microchamber and RNA is automatically extracted and purified. A simple but effective procedure, though is the result of know-how which took many years of research.

The principle of how it functions is quite easy to understand. The side of the chip into which the cell solution is filled, has two electrodes arranged a short distance from each other. Alternating current warms up the solution, causing the cells to burst. The neighbouring chip area consists of a square gel located between two DC electrodes. As RNAs are strongly negatively charged, they are pulled to the other side in a process known as electrophoresis. The rest of the cellular content remains in the same place. The short time between cell lysis and electrophoretic purification separates the RNAs from RNAse enzymes which would normally degenerate the RNAs. During electrophoresis, larger RNAs (for example ribosomal RNAs) get caught in the pores of the gel; only the small RNAs can pass through. They are dissolved in a special solution and are now available in a purified form. However, the small size of the chip gave Vulto and his colleagues a couple of problems to solve.

Bubble-expulsion structures remove electrolytic gas bubbles (left) and the phaseguides enable the controlled filling of the chip. © Dr. Paul Vulto

Gas bubbles are one of the problems the researchers had to overcome. The gas bubbles are created during electrophoresis with DC, which cleaves the water into hydrogen and oxygen. In addition, it is very difficult to fill a microchamber without bubbles occurring due to the scale of the chip which leads to strong capillary forces that allow air to be drawn in. Another problem is that it is impossible to cast such a small gel reproducibly in the desired square shape. In order to remove the bubbles created by the current applied, Vulto developed special microscopic expulsion structures to remove the electrolytic gas bubbles through capillary pressure from the chip. In order to make sure that the fluids and the gel spread evenly in the chambers, Vulto developed specific barriers (phaseguides) which guide the fluids.

Everything on one chip?

“We have compared our chip with a standard laboratory method and have been able to show that our method requires one thousand times fewer cells for extracting the same amount of RNA,” said Vulto. This efficiency might be of particular interest for pharmaceutical companies, as cell material is very expensive. The Swiss company Ayanda Biosystems GmbH has already purchased a licence for the biochip and is currently developing an automated method to test anti-cancer substances in a parallel fashion. Automation is simple because it is only necessary to programme a pipetting robot to fill the chips with cell samples; the rest is done by the chip alone.

In principle, the technology is suitable for other applications. In cooperation with the National Diagnostics Centre in Galway, Ireland, Vulto has been involved in the development of a diagnostic test for hospital bacteria. Another project, funded by the German government, focuses on using the technology for the detection of pathogens that might become important in the food industry or in bioterrorism. In cooperation with the Robert Koch Institute and the Department of Virology at the University of Göttingen, Vulto’s colleagues at the IMTEK are currently testing whether the system is also suitable for the extraction and purification of DNA and proteins. This might be very useful for the detection of viruses.

Vulto envisages that further work steps can be included on the chip, for example a PCR test to directly determine the isolated RNAs or DNAs. If the researchers succeed, then this might turn a vision into reality that has already given the name to this type of miniaturised technology: lab-on-a-chip.

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/more-effective-extraction-of-small-rnas