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

Abstract

Abstract Background 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. Results High 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. Conclusions The 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.

Keywords

• RNA-seq
• rRNA depletion
• HISAT
• Degraded FFPE sample