Transcriptomic profiling of human skin biopsies in the clinical trial setting: A protocol for high quality RNA extraction from skin tumours

Introduction

Skin has evolved as a protective barrier against environmental stresses such as physical damage, ultraviolet radiation exposure and pathogenic infections. It is a complex tissue, consisting of many cell types, including keratinocytes, immune cells, melanocytes, fibroblasts, adipocytes, nerve fibres, smooth muscle and endothelial cells (Cole et al., 2001). Skin diseases may involve any of these constituent cells, and transcriptomic profiling of diseased human skin can inform understanding of disease pathogenesis. The accessible nature of the skin, unlike tissues such as the brain or liver, and the low patient impact of skin biopsy, make the use of transcriptomics in human skin particularly attractive. Whole punch biopsies have been studied using transcriptome-wide approaches, giving insights into the inflammatory skin disease psoriasis (Zolotarenko et al., 2016).

Reliably obtaining high quality RNA of sufficient quantity from small samples typical of those derived from punch biopsies is, however, problematic. The reasons for this are diverse and include suboptimal tissue disruption and endogenous RNase activity, resulting in skin being a tissue that is amongst the most challenging to obtain intact RNA from (Walker et al., 2016)(Kaufmann et al., 1980). RNA is also a highly unstable molecule. Once extracted from cells or tissues it has a very short half-life and is easily degraded (Brooks, 1998; Tan & Yiap, 2009). RNA is chemically unstable due to the presence of a hydroxyl group at the 2’ and thus is highly susceptible to hydrolysis by ribonuclease enzymes. The hydrolysis or cleavage of RNA can also occur spontaneously, without the presence of a catalyst or enzyme (Elliott & Ladomery, 2011). Together, these challenges may discourage the inclusion of transcriptomics in clinical trials involving human skin, where such information may offer mechanistic insights.

Methods of RNA extraction from human skin have been evolving. Early approaches required large amounts of tissue, and were not amenable to medium/high throughput lysis, as each sample had to be disrupted in liquid nitrogen (Hipfel et al., 1998; Kaufmann et al., 1980). Whole punch biopsies of the skin have been successfully disrupted by low throughput devices, such as the Kinematica Polytron 1300D homogenizer and FastPrep120 bead beater (Berglund et al., 2007). 4 mm skin biopsies have also been subjected to ammonium thiocyanate-induced dermo-epidermal separation and subsequently homogenized using bead-based lysis (Clemmensen et al., 2009); however the RNA integrity may have been suboptimal due to tissue processing. Importantly none of these approaches allow for histological assessment of the skin sample, which is relevant in interpreting sequencing results of diseased skin. Laser capture microdissection is an alternative method with histological information, with the limitations being cost of laser capture microscopy equipment, small amount of RNA obtained, and RINs that may be lower (circa 7.0) (Butler et al., 2016). An additional amplification stage is also often required when starting from small amounts of RNA during library generation, which may introduce an element of bias (Jackson et al., 2014).

Here we report an efficient method of RNA extraction from skin tumours obtained using punch biopsies under local anaesthetic in a clinical trial involving patients with an inherited skin tumour syndrome. We also validated this method in normal human skin biopsies. Downstream analysis demonstrated high yield and RNA integrity, allowing for transcriptomic profiling using RNA sequencing.

Methods

Sample processing

A full working protocol is detailed in Supplementary File 1, and the key steps are outlined here. Standard precautions to prevent contamination with RNAses were employed. The sample was removed from the freezer and transferred on dry ice to the cryostat. The sample identifier was used to label slides and preweighed lysis tubes, which were supplied prefilled with beads (Precellys). Skin punch biopsy samples of tumours (4–6 mm diameter) were mounted on a cork piece using cutting compound (OCT), and then serially sectioned (Figure 1). Each skin biopsy was cryosectioned at two levels and ten 30-micron curls were taken from each level for RNA extraction; two were taken from each biopsy. Sections were then taken for histology and applied to a standard slide, and subject to haematoxylin and eosin staining. Following cryosectioning, material was immediately placed into the bead lysis tube, weighed again and then cold lysis buffer (RLT) containing beta-mecaptoethanol was added (Qiagen RNeasy Micro Kit No 74004). The tube was frozen on dry ice and then returned to the -80°C freezer until RNA extraction was performed.

Figure 1. A method for extracting high quality RNA from small skin biopsy samples which is suitable for RNA sequencing.

(a) Diagram indicating key steps in the work flow, and the need to keep samples cold throughout. (A) Human skin punch biopsy (B) Freezing of sample within 30 seconds. (C–D) Cryostat sectioning of punch biopsy and curls obtained. (E–F) Addition of lysis buffer to single use bead tube, followed by homogenisation in the bead-based lysis machine where up to 24 samples can be processed at once. (G) Silica spin column based RNA extraction performed at 4 degrees. (b) Microfluidic gel analysis of total RNA demonstrates distinct 18S and 28S ribosomal bands, consistent with the high RNA integrity scores (c) demonstrated across samples. (d) FASTQc assessment of reads indicate high quality reads in libraries developed from this dataset.

Results

Discussion

Due to its high sensitivity and resolution, RNA sequencing is a powerful tool for investigation of skin disease. It can provide mechanistic insights behind disease pathogenesis, which may offer prognostic information, prediction of response to treatments and the potential for developing novel therapies (Gilmore, 2013). To overcome the inherent difficulties of obtaining high integrity RNA, there are in general, four crucial steps required during extraction: cells or tissues should be completely homogenised; nucleoprotein complexes should be disrupted; RNase should be inactivated; contamination including carbohydrate, lipids, protein and other nucleic acid should be avoided (Buckingham & Flaws, 2007; Doyle & Doyle, 1990).

We describe a novel method, incorporating cryostat sectioning, resulting in partial disruption of the tissue, whilst simultaneously allowing us to take 8 μm sections for histological assessment. We used bead-based tubes that allowed for up to 24 samples to be processed simultaneously. Importantly, we were able to work with small amounts of skin, which were robustly disrupted using the bead-based lysis system we employed. This prevented carryover of incompletely homogenised material and obstruction of spin columns used for RNA extraction. The bead-based lysis system we employed had a range of disruption settings and after optimisation with a range of bead sizes, we chose a medium bead mix (CK28 Mix), and lysis of 20 seconds at 5500 rpm in lysis buffer as this gave optimal RNA extraction and RINs.

After skin homogenisation, there are several established methods for RNA extraction. Phenol/chloroform extraction, one of the commonly used techniques, has potential for phenol contamination of the samples, which is often reported (Oñate-Sánchez & Vicente-Carbajosa, 2008). Therefore, we employed a silica column-based RNA extraction methodology (Sellin Jeffries et al., 2014). This approach suited our protocol, which required small volumes of lysis buffer, and elution of RNA performed in 14ul of water. This typically resulted in highly concentrated samples and thus satisfied the requirements of most RNA library preparation kits for next generation sequencing.

Given RNA’s sensitivity to temperature, we developed a protocol where the skin sample is kept as cold as possible throughout. The tumour samples were first snap frozen in liquid nitrogen, and then cryosectioned at a temperature of -20°C. During rapid homogenisation in our protocol, the tumour material was exposed to ambient temperatures for only a few minutes. This was immediately followed by RNA extraction in a precooled centrifuge at 4°C. Spin columns were incubated on wet ice between centrifugation steps and eluted RNA was immediately frozen after RNA extraction was complete.

In summary, we developed a method for efficient extraction of RNA from small cryosectioned pieces of human skin with histological data from adjacent tissue sections. We validated this method in human skin tumours samples in a clinical trial setting. This protocol could be utilised in healthy and diseased skin to improve mechanistic understanding in a range of human skin disease and cancer.

Data availability

FASTQ files (controls [n=3] and tumours [n=3] used to generate validation signatures), sample description files and uncropped gel images are available at Open Science Framework: http://doi.org/10.17605/OSF.IO/5YX96 (Rajan, 2018).

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).