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

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. With the development of massive parallel sequencing, RNA expression profiles have become an important source of new biomarkers with potential values in cancer metastasis and disease prognosis. Standard practice in tissue fixing and paraffin-embedding has little impact on the expression analysis of RNA samples, which makes archived FFPE samples valuable and retrospective studies feasible. But it is still difficult for some FFPE samples which have low quality or low amount of RNA to be used in the RNA expression study. 

Methods: We compared four FFPE RNA library preparation kits (KAPA, TaKaRa, QIAGEN and Vazyme) based on two principles of rRNA depletion, with total RNA from FFPE samples and paired FF samples using two bioinformatics analysis methods. Furthermore, we tried to construct RNA libraries with the two kits allowing for degraded low-input RNA using clinical FFPE samples of different quality. HISAT was used to analyze the data of all the libraries. 

Results: Our results demonstrated that high concordance in transcript quantifications were shown between FF and FFPE samples using the same kit and more transcripts could be obtained by rRNA depletion after reverse transcript reaction. The gene expression profiling 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 higher residual rRNA and lower unique mapping ratio in the libraries. 

Conclusions: These results indicated that most of clinical FFPE samples could be used in the RNA profiling study. 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.