TY - JOUR
T1 - Effect of formalin-fixation and paraffin-embedded tissue storage times on RNAscope in situ hybridization signal amplification
AU - Colburn, Megan E.
AU - Delaney, Martha A.
AU - Anchor, Gretchen C.
AU - Terio, Karen A.
N1 - Publisher Copyright:
© 2024 The Author(s).
PY - 2024/7
Y1 - 2024/7
N2 - RNAscope in situ hybridization (ISH) detects target RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. Protocols suggest that prolonged FFPE storage and formalin fixation may impact signal detection, potentially limiting the utility of RNAscope ISH in retrospective studies. To develop parameters for RNAscope use with archived specimens, we evaluated the effect of formalin-fixation time by measuring the signal of a reference gene (16srRNA) in selected tissues fixed in 10% neutral-buffered formalin for 1, 2, 3, 5, 7, 10, 14, 21, 28, 60, 90, 180, and 270 d. The signal intensity and percent area of signal decreased after 180 d. Tissues had detectable signal at 180 d but not at 270 d of formalin fixation. To assess target detection in paraffin blocks, we qualitatively compared the signal of canine distemper virus (CDV) antigen via immunohistochemistry and CDV RNA via RNAscope ISH in replicate sections from blocks stored at room temperature for 6 mo, 1, 3, 6, 8, 11, 13, and 15 y; RNA was detected in FFPE tissues stored for up to 15 y. Our results demonstrate that RNAscope ISH can detect targets in tissues with prolonged paraffin storage intervals and formalin-fixation times.
AB - RNAscope in situ hybridization (ISH) detects target RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. Protocols suggest that prolonged FFPE storage and formalin fixation may impact signal detection, potentially limiting the utility of RNAscope ISH in retrospective studies. To develop parameters for RNAscope use with archived specimens, we evaluated the effect of formalin-fixation time by measuring the signal of a reference gene (16srRNA) in selected tissues fixed in 10% neutral-buffered formalin for 1, 2, 3, 5, 7, 10, 14, 21, 28, 60, 90, 180, and 270 d. The signal intensity and percent area of signal decreased after 180 d. Tissues had detectable signal at 180 d but not at 270 d of formalin fixation. To assess target detection in paraffin blocks, we qualitatively compared the signal of canine distemper virus (CDV) antigen via immunohistochemistry and CDV RNA via RNAscope ISH in replicate sections from blocks stored at room temperature for 6 mo, 1, 3, 6, 8, 11, 13, and 15 y; RNA was detected in FFPE tissues stored for up to 15 y. Our results demonstrate that RNAscope ISH can detect targets in tissues with prolonged paraffin storage intervals and formalin-fixation times.
KW - RNAscope
KW - archival tissue
KW - in situ hybridization
KW - retrospective
KW - viral detection
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U2 - 10.1177/10406387241245777
DO - 10.1177/10406387241245777
M3 - Article
C2 - 38650110
AN - SCOPUS:85191333606
SN - 1040-6387
VL - 36
SP - 498
EP - 505
JO - Journal of Veterinary Diagnostic Investigation
JF - Journal of Veterinary Diagnostic Investigation
IS - 4
ER -