Magnetic Composite Particles In The Mrna Purification Applications

Superparamagnetic composite particles are composed of ferrofluid (such as iron oxide, Fe, Co, Ni nanoparticles) and inorganic/organic materials. Antibodies, enzymes, nucleic acids/oligonucleotides, molecular probes or medicine can be immobilized on magnetic particle surface by physical adsorption or covalently interaction between the functional groups (such as amino, carboxyl, thiol) on magnetic particles and biomolecules. Based on the advantages of magnetic separation and the immobilization of biomolecules, magnetic composite particles have been widely applied in the field of cell sorting, enzyme immobilization, immunossay, drug delivery and biomolecule purification.mRNA plays an important role in gene analysis, coupled with oligo(dT)₂₀, magnetic particles have been proved as a ideal carrier in purification of mRNA. The method has advantages such as rapid performance, easy operation, good reproducibility and high purity of mRNA obtained with this method. The mRNA purification kits using magnetic particles have been commercialized in USA, Germany, etc. However, these products are much expensive compared with the traditional method using column coupled with oligo(dT)₂₀. Herein, we developed a mRNA isolation method with different kinds of magnetic particles: the details of immobilization oligo(dT)2o on particle surface, the process and results for mRNA purification from animal tissues and plant tissues are described.Silanized magnetic particles with -NH₂ groups on their surface were activated by 1,4-phenylene diisothiocyanate (PDITC) and the NH₂-labeled oligo(dT)₂₀ were immobilized on magnetic particle surface using another isothiocyanate group in PDITC. The immobilization capability of NH₂-labeled oligo(dT)₂₀ was in the range of 0.85 nmol～0.9 nmol per miligram of magnetic composite particles. The total RNA was firstly isolated from liver, kidney and spleen of rat using traditional method, then the magnetic partcles labelled with oligo(dT)2o was used to isolate mRNA. 2.26μg (liver), 2.45μg (kidney) , 1.77μg (spleen) of mRNA was obtained from 100μg total RNA separately using magnetic particles; In the same manner, 0.93μg (Trifolium repens),1.58μg (quihouicarr) of mRNA was also obtained from 100μg total RNA of plants respectively. All of the ratios of OD₂₆₀/OD₂₈₀ are between 1.8 and 2.0, and the agarose electrophoresis result shows that the mRNA is smear. The mRNA from rat liver was used as template for amplification of 6-actin gene via reverse transcription-polymerase chain reaction (RT-PCR) and the agarose electrophoresis result shows that the target DNA molecule with 240 bp was synthesized using purified mRNA, which indicated that the mRNA purified is integrity.The mRNA isolation from animal tissue directly and purification of mRNA from total RNA using streptavidin coated magnetic composite particles (GoldMag) were also discussed in this thesis.