Tape Strips in Dermatology Research

A. J. Hughes; S. S. Tawfik; K. P. Baruah; E. A. O'Toole; R. F. L. O'Shaughnessy

Disclosures

The British Journal of Dermatology. 2021;185(1):26-35. 

In This Article

What is the Best Protocol?

Extraction protocols vary depending on the epidermal component studied, type of dermatosis and method of analysis. Many variables affect the cohesion of the SC and subsequent yield of protein from TS, which are summarized in Table 3. The type of dermatosis and activity of skin disease affect protein yield.[18,50,54] Lesional AE has increased intracorneocyte cohesion[58] and a subsequent lower yield of protein from TS than nonlesional skin;[50] however, the barrier defect in lesional AE leads to serum 'leakiness'[11] and an increase in the proportion of soluble protein.[50] Lesional psoriasis is hyperkeratotic and yields more protein than nonlesional skin.[17,54] Some variables, such as skin hydration, cannot be standardized, so the data must be normalized. To do this, most studies quantified the total soluble protein per TS sample using an assay to ensure equal protein loading for each sample.[37,59–61] For qPCR, a housekeeping gene is used to normalize gene expression.[9,62]

Protein extraction regimens require mechanical disruption (mechanical shaker,[55] cell scraper,[47] sonication[61] and/or chemical disruption, such as methanol)[47] to remove proteins from the TS. Propylene glycol was added by one author to hydrate keratinocytes and prevent ice crystals forming during sonication.[57] Hendrix et al. found that 60 min of sonication led to lower detection of certain biomarkers and recommended 30 min of sonication to avoid denaturing proteins.[57] Clausen et al. found that 10 min of sonication with phosphate-buffered saline (PBS) led to a 50–90% reduction of protein remaining on TS, with no significant difference in protein yield between 10 and 15 min of sonication.[50] Mechanical/chemical disruption is sufficient to study soluble proteins, but if an insoluble protein is studied (and 85% of protein in the SC is insoluble),[10] these must be solubilized first. Extraction buffers containing reducing agents [e.g. dithiothreitol (DTT)],[61] surfactants (e.g. Tween-20)[44] or bases (e.g. sodium hydroxide)[63] facilitate this. Typical extraction buffers contain between one and three of those ingredients[50,61] and are added at the mechanical disruption stage.[57] Strong extraction buffers yield more protein, but may harm fragile proteins.[50] Higher SDS concentrations interfere with ELISA or protein assays.[57] If samples are used for MS analysis, buffers must be compatible with the machinery[46] and additional clean-up may be necessary to remove the TS polymer and adhesive, which can also interfere with MS.[40] Some studies include a protease inhibitor,[61] although Clausen et al. reported no benefit when studying soluble proteins.[50]

Few studies have directly compared different extraction buffers or protocols. Hendrix et al. analysed multiple variables to determine the optimum extraction protocol for protein extraction from four TS taken from healthy volunteers.[57] The endpoint was the detection of a panel of biomarkers (mostly proteins) representing a range of molecular weights and water solubilities using a luminex kit.[57] The study found no single set of extraction conditions that maximized recovery of all biomarkers, but overall recommended a buffer composed of 0·2% SDS in PBS, a lower extraction temperature (7 ± 3 °C) and 30 min sonication.[57]

RNA is typically extracted using commercial extraction kits, which use a lysis buffer containing guanidinium thiocyanate to denature proteins while preserving RNA, followed by centrifugation in silicone spin columns to separate RNA from DNA and proteins. Four studies used a buffer that also contained phenol.[2,5,9,55] This has the advantage of allowing phase separation of proteins and RNA, although phenol can reduce RNA integrity.[8] TS samples are usually stored at −80 °C following collection.[5,9] Some studies scraped freshly taken TS directly into the lysis buffer before freezing.[43] One study demonstrated no benefit from freshly harvested TS compared with 10 days of storage at −80 °C.[64] Rubber cell scrapers,[43] sonication,[62] pressure cycling technology[8] and vigorous shaking[55] have been used with the lysis buffer to aid separation of RNA from the adhesive. One study anecdotally reported higher RNA yield from pressure cycling technology over sonication.[8] Another used an additional proteinase K digestion step.[62] RNA yields are typically low, so linear whole-transcriptome amplification is usually necessary following reverse transcription.[55] cDNA is then quantified using qPCR,[9] RNA-seq[43] or microarrays.[62]

Comments

3090D553-9492-4563-8681-AD288FA52ACE

processing....