Extraction of high-qualityrna from tumours tissues using mechanical homogenisation method for downstream application / 国际药学研究杂志

ABSTRACT

RNA quality and quantity are crucial factors in ensuring the accuracy of RNA-based downstream applications such as the northern blotting, qPCR, transcriptome and microarray analyses. Bead beating is an effective mechanical lysis method that candisrupt a wide range of biological samples including microorganisms, plants, animals and human tissues. In this experiment, a MINI bead beater, TRIzol™ reagent and zirconium beads were used to demonstrate the bead-beating method in extracting high-quality total RNA from mouse tumour tissues from four different anatomies. The tumour tissues were isolated from the Cdkn2a-/-, Tyr-HRASG12Vmouse model of melanoma that develops spontaneous melanomas. The extracted total RNA was measured through absorbance reading to verify the purity and yield of the product. Whilst, the intactness of the final product was examined using ethidium bromide stained agarose gel electrophoresis. To obtain an overall reflection of the RNA quality, extracted RNAs were subjected to qRT-PCR to evaluate endogenous mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH)gene expressions. The results of the experiment further affirmed that the application of bead beater along with TRIzol™ reagent and zirconium beads has the potential of extracting high-quality RNA from tumour tissues without affecting the yield, purity, integrity and functionality of the RNA.