A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples

Xiaojing Lin,Junyan Hou,Jun Zhao,Lihong Qiu,Weizhi Chen,Xue Song

doi:10.1186/s12864-019-6166-3

Abstract

BackgroundFormalin-fixed and paraffin-embedded (FFPE) blocks held in clinical laboratories are an invaluable resource for clinical research, especially in the era of personalized medicine. It is important to accurately quantitate gene expression with degraded and small amounts of total RNA from FFPE materials.ResultsHigh concordance in transcript quantifications were shown between FF and FFPE samples using the same kit. The gene expression using the TaKaRa kit showed a difference with other kits, which may be due to the different principle of rRNA depletion or the amount of input total RNA. For seriously degraded RNA from FFPE samples, libraries could be constructed with as low as 50 ng of total RNA, although there was residual rRNA in the libraries. Data analysis with HISAT demonstrated that the unique mapping ratio, percentage of exons in unique mapping reads and number of detected genes decreased along with the decreasing quality of input RNA.ConclusionsThe method of RNA library construction with rRNA depletion can be used for clinical FFPE samples. For degraded and low-input RNA samples, it is still possible to obtain repeatable RNA expression profiling but with a low unique mapping ratio and high residual rRNA.

Highlights

Formalin-fixed and paraffin-embedded (FFPE) blocks held in clinical laboratories are an invaluable resource for clinical research, especially in the era of personalized medicine
Residual Ribosome RNA (rRNA) in the TaKaRa library was the highest and had the least clean data, which was due to the removal of ribosomal Complementary DNA (cDNA) after cDNA synthesis using probes specific to mammalian rRNA
Previous research [17, 18] and the results of this study showed that FFPE RNA sequencing (RNA-seq) provided reliable gene expression data, comparable to that obtained from fresh frozen tissue with the method of rRNA depletion

Summary

Introduction

Formalin-fixed and paraffin-embedded (FFPE) blocks held in clinical laboratories are an invaluable resource for clinical research, especially in the era of personalized medicine. It is important to accurately quantitate gene expression with degraded and small amounts of total RNA from FFPE materials. Lin et al BMC Genomics (2019) 20:831 without the 3′poly (A) tail; recent studies suggest that certain functionally important mRNAs are non-poly (A) RNAs [7]. Capturing the 3′poly (A) tail is not a compatible method, especially when the starting materials are from FFPE samples. Another method for RNA-seq of FFPE samples is cDNA hybrid capture using a whole exome DNA probe to hybridize to the total RNA library. The yield of on-exon data was increased significantly due to the cDNA-capture, while the accuracy of quantitated gene expression was decreased [8, 9]. The signals of low gene expression might be missed by decreased uniformity of the exome probe

Methods

Results

Discussion

Conclusion