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Optimization of izolation technique utilized for preparation of template material suitable for gene expression analysis of mesenchymal stromal cells from umbilical cord tissue
NOVÁKOVÁ, Zora
Ribonucleic acid (RNA) is the main component of the regulation of gene expression in the cell. As the development of techniques of molecular biology proceeds, RNA becomes an important tool for the gene expression analysis in research as well as diagnostic approaches. In contrast to deoxyribonucleic acid, RNA is very unstable and its isolation is tricky. RNA used for the gene expression analysis should fulfill several re foquirements focused especially on its purity. The isolation techniques should ensure effective separation of RNA from other cell components and chemicals used in the process. Currently, several methodologies are employed for the RNA isolation. The most common isolation is guanidine thiocyanate-phenol-chloroform combination called TRIzol or TRI Reagent and the extraction on solid phase so-called the column extraction. This study shows basic knowledge about macromolecule RNA and describes methodologies of RNA extraction from biological material. Spectrophotometry is very common technique used in laboratories. Thus the spectrophotometer is considered according to its speed, accuracy and simplicity of utilization. New improved devices reveal higher accuracy and lower requirements for the sample volume and make huge analyses much easier and more accurate. Getting knowledge about the principle and construction of the device we can improve utilization of the device and interpret the data correctly. In this study basic physical laws and principles concerning electromagnetic radiation and spectrophotometry are described, moreover, the basic model of spectrophotometer and critical parameters for nucleic acid quantification are shown. The aim of the study was to find the methodic approach for the isolation of unlimited amount of RNA of high quality suitable for high-throughput gene expression analysis of mesenchymal stromal cells. In the study several isolation techniques were compared to gain RNA suitable for reverse transcription reaction. These include isolation with guanidine thiocyanate-phenol-chloroform combination and column extraction. In total, yield and purity of RNA isolated by seven kits was compared in relation to the amount of cells used. The study involved comparison of two spectrophotometric devices NanoDrop ND-1000 and BioPhotometer Eppendorf. The accuracy of measurement of the concentration and the purity of RNA, speed, sample volume requirement and the weight of data acquired was compared between tested devices. A set of cell samples containing various number of mesenchymal stromal cells and commercially available RNA were used for testing purposes. The results of the study showed that in case of small cell number samples RNA isolated by column extraction is of higher quality than that isolated by TRI Reagent. From column-based kits the ZR RNA MicroPrep gave the best results the highest yield and purity of isolated RNA. From the spectrophotometric devices tested NanoDrop revealed smaller deviation of measurement, shorter time required for measurement, smaller sample volume requirement and higher weight of acquired data. In summary, ZR RNA MicroPrep and NanoDrop spectrophotometer were selected for preparation of RNA samples suitable for gene expression analysis.
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