Projects / Programmes
Implementation of ion chromatography-tandem mass spectrometry in the analytics of short-chained RNA molecules: development of methodology and analytical protocols
| Code |
Science |
Field |
Subfield |
| 4.03.07 |
Biotechnical sciences |
Plant production |
Technology of vegetal origin food |
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
| Code |
Science |
Field |
| 4.04 |
Agricultural and Veterinary Sciences |
Agricultural biotechnology |
ESI ionization, fungal culture, ion chromatography, mass spectrometry, miRNA, plant culture, separation techniques, short-chained RNA, siRNA, tertiary structure, transcriptomics
Researchers (1)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
24445 |
PhD Mitja Križman |
Chemistry |
Head |
2010 - 2012 |
150 |
Organisations (1)
| no. |
Code |
Research organisation |
City |
Registration number |
No. of publicationsNo. of publications |
| 1. |
0104 |
National Institute of Chemistry |
Ljubljana |
5051592000 |
21,019 |
Abstract
The project is focused in the transcriptomics field. During the applied project, new analytical procedures will be developed, based on ion chromatography hyphenated with tandem mass spectrometry for structure (sequence) and nucleotide content determination in short-chained RNA molecules from biological samples.
We will develop an ion-exchange separation system (combination of eluent, chromatographic column and other parameters) able to effectively separate RNA molecules up to 30 nucleotides in length. A more uniform analyte distribution within the separation is expected, compared to reversed-phase separations, due to a different separation mechanism (distribution of ionic charge). The best separation performance in terms of selectivity is expected with columns having a high density of functional groups. Along with a careful choice of other conditions (e.g. mobile phase pH and ionic strength, separation temperature etc.) a discrimination between analytes based on their nucleotide sequence shall be obtained, at least to some extent (due to the differences in RNA secondary/tertiary structure). Most likely, for obtaining an adequate sensitivity (besides selectivity), gradient elution separation will be employed. If necessary, a post-column desalter (suppressor) will also be used for matching the mobile phase composition with mass spectrometric (MS) detection.
Mass spectrometric detection will be based on electrospray (ESI) ionization. The choice of ionization conditions will be crucial, since an excessive RNA ionization shall be prevented in order to exploit the instrumental performance of a quadrupole mass spectrometer. Under optimal ESI ionization conditions the MS detection will enable a selective and sensitive RNA determination, even in complex sample matrices, i.e. cell or tissue culture isolates. Combined with tandem MS detection, RNA characterization in terms of nucleotide composition will also be possible, owing to MS fragmentation.
Several isolates from hop (Humulus lupulus) cultures and phytopathogenic fungal (Verticillium albo-atrum) cultures will be analyzed for their short-chained RNA composition. Cultures will be systematically exposed to various stress conditions (e.g. salinity, temperature etc.) and their RNA composition will be compared. We expect to obtain data on certain RNA molecules as potential stress indicators for these organisms. By understanding the occurrence of characteristic RNA molecules, a better insight into physiological conditions of plant (fungal) culture will be achieved. Consequently, the development of a better approach for in vitro cultivation and a better understanding of cellular mechanisms will be gained.
The effectiveness of a given analytical method depends substantially on sample preparation procedure. Hence, a part of our activities will be dedicated to this topic. At the very start, current protocols for short-chained RNA extraction will be employed and checked for their suitability for IC-MS use. In the case incompatibility of current protocols with MS detection should arise, adaptation of current or the development of new protocols for short-chained RNA extraction will take place.
The field of transcriptomics is quickly expanding with a steady acquisition of scientific discoveries and consequently scientific publications as well, due to its importance within biosciences. Unfortunately, the development of suitable analytical tools is not withstanding the pace of scientific discoveries. We believe the implementation of IC-MS technique will provide a more efficient tool for qualitative and quantitative short-chained RNA analysis.
Significance for science
The developed procedures and the knowledge gained during the project are useful in different scientific disciplines for analyses of short-chained RNA molecules (biochemistry, pharmacy, agronomy, veterinary and human medicine). In comparison to the established molecular genetic techniques, these procedures represent a faster and more inexpensive alternative. It should be emphasized that the development of these new procedures was not primarily intended as a replacement of existing analytical protocols. Rather, it is intended as complementary tool, an alternative and/or an upgrade of existing procedures, especially in screening tests and in gathering potential markers where simplicity of sample preparation is preferred, yet still without deleterious sample matrix effects. The usefulness of RNA separation, detection and sample preparation procedures is thus quite broad: from research in the agronomy field to clinical tests. At the same time, the possibility of using LC-MS equipment instead of hardly accessible IC-MS counterparts for such analyses opens more opportunities for implementing these procedures in other laboratories. The procedure for DNA nucleotide composition analysis (with gas chromatography) is additionally interesting for assessment of organism's oxidation stress level. The procedures' individual segments (separation, detection, sample preparation) are also individually useful and transferrable to other similar applications.
Significance for the country
In the near future, pharmaceutical industry will commercialize drugs based on short-chained RNA molecules (based on the number of relative patents), thus the utility of these analytical procedures will be useful in the development and quality control of these pharma products. Consequentially, it can be expected that the already good collaboration between the National Institute of Chemistry and the pharmaceutical industry will be further strengthened. This methodology has also its place in clinical diagnostics where it could cut down the costs for screening tests for some diseases, as well as in agriculture and in veterinary medicine. In the field of clinical diagnostics, there is also the possibility to conduct oxidation stress tests (private practice or within established health institutions). Last but not least, the new concept for a mobile phase desalter opens up the opportunity for an entrepreneurship initiative, in producing and selling devices with a high added value.
Most important scientific results
Annual report
2010,
2011,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Annual report
2010,
2011,
final report,
complete report on dLib.si
