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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.
BMC Genomics 2019; 20(1):831BG

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.

Authors+Show Affiliations

Department of Thoracic Surgery, Cancer Hospital Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.Department of Thoracic Surgery, Cancer Hospital Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.Department of Thoracic Surgery, Cancer Hospital Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.Department of Thoracic Surgery, Cancer Hospital Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.Department of Thoracic Surgery, Cancer Hospital Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.Genecast Precision Medicine Technology Institute, Room 903-908, Health work, Huayuan North Road 35, Haidian District, Beijing, 100191, China. drzhaojun@126.com.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31703614

Citation

Lin, Xiaojing, et al. "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." BMC Genomics, vol. 20, no. 1, 2019, p. 831.
Lin X, Qiu L, Song X, et al. 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. BMC Genomics. 2019;20(1):831.
Lin, X., Qiu, L., Song, X., Hou, J., Chen, W., & Zhao, J. (2019). 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. BMC Genomics, 20(1), p. 831. doi:10.1186/s12864-019-6166-3.
Lin X, et al. 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. BMC Genomics. 2019 Nov 8;20(1):831. PubMed PMID: 31703614.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Lin,Xiaojing, AU - Qiu,Lihong, AU - Song,Xue, AU - Hou,Junyan, AU - Chen,Weizhi, AU - Zhao,Jun, Y1 - 2019/11/08/ PY - 2019/02/03/received PY - 2019/10/02/accepted PY - 2019/11/10/entrez PY - 2019/11/11/pubmed PY - 2019/11/11/medline KW - Degraded FFPE sample KW - HISAT KW - RNA-seq KW - rRNA depletion SP - 831 EP - 831 JF - BMC genomics JO - BMC Genomics VL - 20 IS - 1 N2 - 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. SN - 1471-2164 UR - https://www.unboundmedicine.com/medline/citation/31703614/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 L2 - https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-019-6166-3 DB - PRIME DP - Unbound Medicine ER -