Increasing mRNA yield from skin samples

Transcriptome research has become increasingly popular to monitor global changes in gene expression. In particular, transcriptome changes between diseases versus healthy states can provide valuable insight into understanding immunoresponse mechanisms in disease states. However, the isolation of high-quality mRNA from skin samples presents numerous challenges, including the toughness of skin, RNase abundance and possibility of degradation resulting from the mechanical disruption process.

Mechanical disruption through dounce homogenisation (glass mortar pestle) has historically been the established protocol for mechanical homogenisation; however, for tough sample type and large-volume sampling, manual dounce homogenisation represents a significant bottleneck. Bead milling, a relatively new technology, is a promising method to overcome these issues. Researchers from the University of Alabama compared these two methods for mRNA extraction with a specific focus on mRNA yields and integrity.

Eight mouse skin samples were obtained from the ear and ventrally. The skin samples were sectioned into 1 cm² samples, separated from fat and kept on ice until homogenisation. They were then diced into fragments, placed in 1 mL of Trizol and homogenised both manually in a dounce homogeniser and by bead beating on an Omni Bead Ruptor 24 in 2 mL bead tubes prefilled with 2.8 mm RNase-/DNase-free ceramic beads. To evaluate if mRNA yields correlate with starting sample quantity, the homogenisation was repeated using 2 x 1 cm² ear and ventral skin samples.

After homogenisation, samples were centrifuged to pellet insoluble material. 30 μL of supernatant was subjected to standard mRNA extraction protocols and OD 260/280 readings were taken to determine mRNA concentration and purity. Based on determined concentrations, approximately 1 μg was mixed with loading buffer and run on a 1.2% non-denaturing gel following heat treatment at 70°C x 1 min then snap cooling on ice. The mRNA purity and integrity of each sample was additionally analysed using an Agilent 2100 BioAnalyzer.

The results indicated that bead milling produced high mRNA yields with good integrity. 280 nm absorbance values indicated that mRNA yields ranged from 26 to 99 μg when bead milling was performed, compared to 9–30 μg achieved through dounce homogenisation, representing an average four-fold increase in mRNA yield. 260/280 nm ratios demonstrate mRNA purity was comparable between both homogenisation techniques; thus, bead milling does not affect sample purity. Electropherograms produced with the Agilent 2100 Bioanalyzer revealed that dounce homogenisation produced samples with high molecular weight contaminant DNA and low molecular weight degradation species, while bead milling homogenisation produced mRNA of high purity and RNA integrity.

The study thus demonstrates the efficacy of using bead mill technology for sample preparation in transcriptomic research.