Identification of an optimal method for extracting RNA from human skin biopsy, using domestic pig as a model system

Abstract

To evaluate skin tissue gene expression patterns correctly, extracting sufficient quantities of good quality RNA is essential. However, RNA extraction from skin tissue is challenging, as the hyaluronic acid-collagen matrix is extremely difficult to homogenize. Although there are multiple ways to extract RNA from skin, there are no comparative studies that identify the most critical steps, e.g. sample collection, storage and homogenization. We analysed the various steps involved in RNA extraction (i.e. biopsy collection as dry biopsy or into nucleotide stabilizing reagents, different storage conditions, enzymatic digestion, stator-rotor and bead motion-based homogenizing combined with column-based RNA purification). We hypothesised that domestic pig skin is applicable as a model for human skin studies. Altogether twenty different workflows were tested on pig skin and the four most promising workflows were tested on human skin samples. The optimal strategy for extracting human skin RNA was to collect, store and homogenize the sample in RLT lysis buffer from the RNeasy Fibrous Tissue Kit combined with beta-mercaptoethanol. Both stator-rotor and bead motion-based homogenizing were found to result in high quality and quantity of extracted RNA. Our results confirmed that domestic pig skin can be successfully used as a model for human skin RNA studies.

Optimization of RNA extraction methods from human metabolic tissue samples of the COMET biobank

“…Manual disruption by syringe/needle can be a good alternative method which provides RNA with comparable quality to GentleMACS Dissociator, but only in case of soft tissues 14 or isolated cells 15 . As also shown for skin biopsies 13 , we observe that RNA yields tend to be higher with GentleMACS Dissociator, but no statistical correlation can be found between biopsy weight and RNA levels, i.e., larger biopsies does not necessarily yield to the highest amounts of RNA. This variation can be due to heterogeneity in cellular composition and transcriptional activity in human tissues/organs, with implications on RNA profiles.…”

“…As compared to FastPrep-24 Instrument, GentleMACS Dissociator seems to be more efficient to dissociate human tissues in one cycle, avoiding repeated lysis cycles and thus preserving RNA integrity. Such a difference was also evidenced on skin biopsies (which are very difficult to dissociate), where GentleMACS Dissociator is the only system allowing a complete homogenization of human skin samples, as compared to FastPrep-24 Instrument 13 . Manual disruption by syringe/needle can be a good alternative method which provides RNA with comparable quality to GentleMACS Dissociator, but only in case of soft tissues 14 or isolated cells 15 .…”

Distinct p63 and p73 Protein Interactions Predict Specific Functions in mRNA Splicing and Polyploidy Control in Epithelia

“…According to the single cell RNA-seq of the human skin, the p73 RNA is under detection threshold [ 55 ]. However, to release single cells, the skin is digested at 37 °C for 2 h, which can lead to RNA degradation, especially for the unstable transcripts [ 58 , 59 ].…”

Reliable and robust droplet digital PCR (ddPCR) and RT-ddPCR protocols for mouse studies

“…Good lysis and homogenization of ventral skin is difficult to achieve with our protocol (Table 1) and may explain the low RNA quality. The adaptation of the existing protocol to mouse skin samples must be done to ensure accurate quantification [17].…”