Session Time: 4:30pm-5:30pm
Presentation Time: 4:55pm-5:00pm
*Purpose: Skin biopsies are currently the gold standard for evaluating the inflammatory status of face allografts. However, “tape stripping” is emerging as a noninvasive technique to monitor autoimmune/auto inflammatory skin diseases. This technique, highly suitable for serial sampling and combined with high sensitive PCR assays, could revolutionize molecular monitoring the face allograft status. Herein, we tested the hypothesis that isolation of total RNA and gene expression profiling are feasible using skin samples collected using tape strips.
*Methods: Tape strips (CuDerm Corporation, Texas) were used to obtain RNA from two face transplant recipients. Each tape strip is comprised of 10 discs. Allograft skin area was marked, cleaned with alcohol, and 8 samples (8 tape strips) were obtained. Each disc (10 discs from one sample) was immersed in 1ml RLT buffer with 100ul β-mercaptoethanol. RNA was isolated from the tape strip skin samples using the RNeasy mini kit. We quantified total RNA using A260/A280 ratio, reverse transcribed RNA into cDNA and pre-amplified cDNA using oligonucleotide primer pairs for a custom panel of mRNAs. We measured absolute levels of mRNA for Keratin 15, MIP1α, and MIP1β, as well as 18S rRNA by preamplification enhanced real time quantitative PCR assays (RT-qPCR assays) using Quant Studio 6. We designed gene specific Taqman primers and probes to amplify and detect gene of interest and a customized BAK amplicon to develop a standard curve for absolute quantification of transcript copies per microgram of total RNA.
*Results: Median quantity of total RNA from the tape strips was 64.48ng. Individual and the median number of total RNA, individual and the median number of the reference gene 18S rRNA, and individual and median number of mRNA copies of Keratin 15, MIP1α, and MIP1β are shown in Figure 1.
*Conclusions: We have demonstrated the feasibility of isolating total RNA from tape strips and quantifying transcript abundance. Further refinement of this technology is ongoing in our laboratory. Tape strip based molecular monitoring of face transplant recipients may offer a noninvasive substitute for allograft biopsies.
To cite this abstract in AMA style:Snopkowski C, Rabbani P, Yang H, Berman Z, Diep G, Li C, Muthukumar T, Ding R, Ceradini D, Suthanthiran M. A Noninvasive Diagnostic Approach for Molecular Monitoring of Face Transplant Recipients [abstract]. Am J Transplant. 2021; 21 (suppl 3). https://atcmeetingabstracts.com/abstract/a-noninvasive-diagnostic-approach-for-molecular-monitoring-of-face-transplant-recipients/. Accessed January 18, 2022.
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