Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Comparison of skin biopsy sample processing and storage methods on high dimensional immune gene expression using the Nanostring nCounter system.

Author(s): Vider J, Croaker A, Cox AJ, Raymond E, Rogers R, Adamson S, Doyle M, O'Brien B, Cripps AW, West NP

Publication: Diagn Pathol, 2020, Vol. 15, Page 57

PubMed ID: 32414387 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of six different preservation methods of skin biopsy specimens on RNA yield, RNA quality, and gene expression analysis. Six 4 mm biopsies were obtained from three healthy patients undergoing abdominoplasty (details not provided). Specimens were preserved by: i) snap freezing in liquid nitrogen and transportation on dry ice; ii) RNAlater for 24 h at room temperature then storage at -80°C; iii) formalin fixation and paraffin embedding (details not provided) and then storage of FFPE blocks at room temperature; iv) Zymo DNA/RNA Shield storage and shipment at room temperature; v) placed in TRIzol and stored at -80°C; vi) 0.15 ml saline without RNAse for 24 h at room temperature then storage at -80°C. Specimens were homogenized using gentle-MACS octo and M tubes. RNA was extracted from liquid nitrogen, saline and RNAlater specimens using the Maxwell RSC simplyRNA Tissue Kit, from FFPE specimens with the RNeasy Mini Kit, from TriZol specimens with the Direct-Zol RNA Kit, and from DNA/RNA shield specimens with the Quick RNA Miniprep Kit. After isolation, RNA was aliquoted and stored at -80°C until analysis. RNA yield and A260/A280 ratios were measured by NanoDrop and RNA Quality Scores (RQSs) were calculated by a bioanalyzer. Gene expression analysis was performed on a NanoString nCounter analysis system using the nCounter PanCancer Immune Profiling panel. Immune gene expression counts were determined using cell-specific gene expression from The Cancer Genome Atlas (TCGA) and those identified as above background (geometric mean of negative controls) were noted as counts <100, 100-1000, and >1000.

   Summary of Findings:

   RNA was extracted from all specimens but yield and quality varied widely among preservation methods. While RNA yields and RQSs were most consistent for FFPE specimens (30.13-68.02 ng/µl and 5.3-5.9, respectively), RQSs were highest for RNAlater specimens and lowest for the saline specimens (8.2 ± 1.15 versus <2). Gene expression counts were comparable for all specimens with the exception of those stored in saline which were below the level of detection. FFPE specimens had the highest mean gene expression counts above background (730), above 1000 (36.75 ± 1.5), and between 100 and 1000 (199.25 ± 9.46) and had the lowest counts below 100 (494 ± 8.28).

   Biospecimens

   • Tissue - Skin

   Preservative Types

   • Formalin
   • RNAlater
   • Other Preservative
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Next generation sequencing
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Snap frozen RNAlater Saline Frozen in TRIzol Zymo DNA/RNA Shield

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