J Breast Cancer.  2017 Dec;20(4):393-399. 10.4048/jbc.2017.20.4.393.

Optimization of RNA Extraction from Formalin-Fixed Paraffin-Embedded Blocks for Targeted Next-Generation Sequencing

Affiliations
  • 1Department of Pathology, Korea University Guro Hospital, Seoul, Korea. idea1@hanmail.net

Abstract

PURPOSE
Breast cancer has a high prevalence in Korea. To achieve personalized therapy for breast cancer, long-term follow-up specimens are needed for next-generation sequencing (NGS) and multigene analysis. Formalin-fixed paraffin-embedded (FFPE) samples are easier to store than fresh frozen (FF) samples. The objective of this study was to optimize RNA extraction from FFPE blocks for NGS.
METHODS
RNA quality from FF and FFPE tissues (n=5), expected RNA amount per unit area, the relationship between archiving time and quantity/quality of FFPE-extracted RNA (n=14), differences in quantitative real-time polymerase chain reaction (qRT-PCR) and NGS results, and comparisons of both techniques with tissue processing at different institutions (n=96) were determined in this study.
RESULTS
The quality of RNA did not show any statistically significant difference between paired FF and FFPE specimens (p=0.49). Analysis of tumor cellularity gave an expected RNA amount of 33.25 ng/mm2. Archiving time affected RNA quality, showing a negative correlation with RNA integrity number and a positive correlation with threshold cycle. However, RNA from samples as old as 10 years showed a 100% success rate in qRT-PCR using short primers, showing that the effect of archiving time can be overcome by proper experiment design. NGS showed a higher success rate than qRT-PCR. Specimens from institution B (n=46), which were often stored in a refrigerator for more than 6 hours and fixed without slicing, showed lower success rates and worse results than specimens from the other institutes.
CONCLUSION
Archived FFPE tissues can be used to extract RNA for NGS if they are properly processed before fixation. The expected amount of RNA per unit size calculated in this study will be useful for other researchers.

Keyword

Breast neoplasms; Estrogens; Receptors; RNA; Sequence analysis

MeSH Terms

Academies and Institutes
Breast Neoplasms
Estrogens
Follow-Up Studies
Humans
Korea
Prevalence
Real-Time Polymerase Chain Reaction
RNA*
Sequence Analysis
Estrogens
RNA

Figure

  • Figure 1 Measurement of tumor cellularity in sample 10_2. (A) Tumor size was measured at 180.1 mm2 using hematoxylin and eosin (H&E) stained sections (×12.5). (B) The total number of cells was determined using an image analyzer (H&E stain, ×100). (C) More than 95% of tumor cells were positive for estrogen receptor (ER)-immunohistochemical (IHC) stain (×100). (D) The number of tumor cells on ER-IHC-stained slides were counted with an image analyzer.

  • Figure 2 Comparison of RNA integrity numbers (RINs) between fresh froze (FF) and formalin-fixed paraffin-embedded (FFPE) specimens.


Reference

1. Jung KW, Won YJ, Kong HJ, Oh CM, Lee DH, Lee JS. Prediction of cancer incidence and mortality in Korea, 2014. Cancer Res Treat. 2014; 46:124–130. PMID: 24851103.
Article
2. Hedegaard J, Thorsen K, Lund MK, Hein AM, Hamilton-Dutoit SJ, Vang S, et al. Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue. PLoS One. 2014; 9:e98187. PMID: 24878701.
Article
3. Lewis F, Maughan NJ, Smith V, Hillan K, Quirke P. Unlocking the archive: gene expression in paraffin-embedded tissue. J Pathol. 2001; 195:66–71. PMID: 11568892.
4. Nechifor-Boilă AC, Loghin A, Vacariu V, Halaţiu VB, Borda A. The storage period of the formalin-fixed paraffin-embedded tumor blocks does not influence the concentration and purity of the isolated DNA in a series of 83 renal and thyroid carcinomas. Rom J Morphol Embryol. 2015; 56(2 Suppl):759–763. PMID: 26429169.
5. Lee JE, Kim JH, Hong EJ, Yoo HS, Nam HY, Park O. National Biobank of Korea: quality control programs of collected-human biospecimens. Osong Public Health Res Perspect. 2012; 3:185–189. PMID: 24159512.
Article
6. Scolnick JA, Dimon M, Wang IC, Huelga SC, Amorese DA. An efficient method for identifying gene fusions by targeted RNA sequencing from fresh frozen and FFPE samples. PLoS One. 2015; 10:e0128916. PMID: 26132974.
Article
7. Ribeiro-Silva A, Zhang H, Jeffrey SS. RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies. BMC Mol Biol. 2007; 8:118. PMID: 18154675.
Article
8. Rupp GM, Locker J. Purification and analysis of RNA from paraffin-embedded tissues. Biotechniques. 1988; 6:56–60. PMID: 2483655.
9. Tsoi DT, Inoue M, Kelly CM, Verma S, Pritchard KI. Cost-effectiveness analysis of recurrence score-guided treatment using a 21-gene assay in early breast cancer. Oncologist. 2010; 15:457–465. PMID: 20421264.
Article
10. Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, et al. A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med. 2004; 351:2817–2826. PMID: 15591335.
Article
11. Wallden B, Storhoff J, Nielsen T, Dowidar N, Schaper C, Ferree S, et al. Development and verification of the PAM50-based Prosigna breast cancer gene signature assay. BMC Med Genomics. 2015; 8:54. PMID: 26297356.
Article
12. Prat A, Bianchini G, Thomas M, Belousov A, Cheang MC, Koehler A, et al. Research-based PAM50 subtype predictor identifies higher responses and improved survival outcomes in HER2-positive breast cancer in the NOAH study. Clin Cancer Res. 2014; 20:511–521. PMID: 24443618.
Article
13. Chung JY, Braunschweig T, Hewitt SM. Optimization of recovery of RNA from formalin-fixed, paraffin-embedded tissue. Diagn Mol Pathol. 2006; 15:229–236. PMID: 17122651.
Article
14. Fleige S, Pfaffl MW. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med. 2006; 27:126–139. PMID: 16469371.
Article
15. Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, et al. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol. 2006; 7:3. PMID: 16448564.
Article
16. Guo Y, Li CI, Ye F, Shyr Y. Evaluation of read count based RNAseq analysis methods. BMC Genomics. 2013; 14(Suppl 8):S2.
Article
17. Rosen PP, Menendez-Botet CJ, Nisselbaum JS, Urban JA, Miké V, Fracchia A, et al. Pathological review of breast lesions analyzed for estrogen receptor protein. Cancer Res. 1975; 35(11 Pt 1):3187–3194. PMID: 171066.
Full Text Links
  • JBC
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr