Endogenous reference RNAs for microRNA quantitation in formalin-fixed, paraffin-embedded lymph node tissue

Katsushige Inada,Toru Shimazui,Akinori Oki,Mitsuo Hori,Hisashi Suzuki,Yoshio Tamaki,Yasushi Okoshi,Tatsuo Iijima,Shingo Ishiguro,Yukiko Cho-Isoda,Hitoaki Saito,Hiroshi Kojima

doi:10.1038/s41598-018-24338-7

Abstract

Lymph node metastasis is one of the most important factors for tumor dissemination. Quantifying microRNA (miRNA) expression using real-time PCR in formalin-fixed, paraffin-embedded (FFPE) lymph node can provide valuable information regarding the biological research for cancer metastasis. However, a universal endogenous reference gene has not been identified in FFPE lymph node. This study aimed to identify suitable endogenous reference genes for miRNA expression analysis in FFPE lymph node. FFPE lymph nodes were obtained from 41 metastatic cancer and from 16 non-cancerous tissues. We selected 10 miRNAs as endogenous reference gene candidates using the global mean method. The stability of candidate genes was assessed by the following four statistical tools: BestKeeper, geNorm, NormFinder, and the comparative ΔCt method. miR-103a was the most stable gene among candidate genes. However, the use of a single miR-103a was not recommended because its stability value exceeded the reference value. Thus, we combined stable genes and investigated the stability and the effect of gene normalization. The combination of miR-24, miR-103a, and let-7a was identified as one of the most stable sets of endogenous reference genes for normalization in FFPE lymph node. This study may provide a basis for miRNA expression analysis in FFPE lymph node tissue.

Highlights

Formalin fixation and paraffin embedding is the universal standard pathological technique for preserving tissue
The top 10 endogenous reference gene candidates selected by the low variability of the ΔCtGlo values were miR-16, miR-21, miR-24, miR-34a, miR-92a, miR-103a, miR-148b, miR-152, miR-191, and let-7a
We presented the use of the mean Ct value of miR-24, miR-103a, and let-7a as a suitable normalization factor for miRNA expression in FFPE lymph node tissue using Quantitative real-time PCR (qRT-PCR)

Summary

Introduction

Formalin fixation and paraffin embedding is the universal standard pathological technique for preserving tissue. MiRNAs regulate post-transcriptional gene expression by binding to the complementary sites of target mRNAs and have important functions in oncogenesis or as tumor suppressor due to their prominent role in cancer pathway regulation[6]. Because of their stability in FFPE tissue, miRNAs can serve as invaluable cancer biomarkers. Quantitative real-time PCR (qRT-PCR) using the threshold cycle (Ct) value is one of the most sensitive methods for miRNA detection and quantification In this method, an endogenous reference gene, which is stably expressed in every sample, regardless of the pathogenesis or diagnosis, is used for normalization. A stable endogenous reference gene should have high and constant level of expression in all FFPE tissue samples

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