Comparison of tumour tissue homogenisation methods: mortar and pestle versus ball mill

Abstract

AbstractBackgroundEfficient genetic material (DNA and RNA) and protein isolation are crucial for obtaining scientifically significant results in biotechnological analytical procedures. DNA mutations, gene expression determination on transcript and protein levels and high-throughput screening are core analyses in cancer studies. The most common tissue homogenisation methods include mortar and pestle usage. This study compares the classic pulverisation method with the nonconventional use of a ball mill.MethodsThe biological material constituted cancerous and unchanged adjacent tissues collected from five patients with head and neck squamous cell carcinoma (HNSCC). Tissues were halved for trituration using both homogenisation methods. The obtained material was used for DNA, RNA, and protein isolation and further PCR, RT-qPCR, and Western-blot analysis.ResultsAfter tissue homogenisation in a ball mill, we found significantly higher DNA concentration than mortar and pestle usage but no significant differences in RNA concentration and DNA and RNA purity ratios. However, the DNA quality assessed by gel electrophoresis and PCR was more excellent in samples ground with mortar and pestle. On the contrary, we demonstrated better RNA quality in ball-milled samples and gene expression analysis using RT-qPCR. We found no significant differences between protein concentration and quality extracted from tissues homogenised with the two compared methods.ConclusionOur results demonstrated that both methods of tissue homogenisation: ball mill versus mortar and pestle, are suitable for human tissue homogenisation to use the DNA and protein in downstream analysis. The ball mill homogenisation is more suitable for RNA extraction and gene expression analysis.