Evaluation and adaptation of a laboratory-based cDNA library preparation protocol for retrospective sequencing of archived microRNAs from up to 35-year-old clinical FFPE specimens

Olivier Loudig, Tao Wang, Qian K. Ye, Juan Lin, Yihong Wang, Andrew Ramnauth, Christina Liu, Azadeh Stark, Dhananjay Chitale, Robert Greenlee, Deborah Multerer, Stacey Honda, Yihe Daida, Heather Spencer Feigelson, Andrew Glass, Fergus J. Couch, Thomas E. Rohan, Iddo Z. Ben-Dov

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.

Original languageEnglish (US)
Article number627
JournalInternational Journal of Molecular Sciences
Volume18
Issue number3
DOIs
StatePublished - Mar 14 2017

Fingerprint

sequencing
paraffins
MicroRNAs
Gene Library
Formaldehyde
Paraffin
Paraffins
cancer
breast
preparation
Carcinoma, Intraductal, Noninfiltrating
evaluation
Breast Neoplasms
RNA
Tissue
Nucleic acids
Nucleic Acids
History
nucleic acids
lesions

Keywords

  • cDNA library preparation
  • Ductal carcinoma in situ
  • Formalin-fixed paraffin-embedded
  • Invasive breast cancer
  • microRNAs
  • Next-generation sequencing

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Computer Science Applications
  • Spectroscopy
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

Cite this

Evaluation and adaptation of a laboratory-based cDNA library preparation protocol for retrospective sequencing of archived microRNAs from up to 35-year-old clinical FFPE specimens. / Loudig, Olivier; Wang, Tao; Ye, Qian K.; Lin, Juan; Wang, Yihong; Ramnauth, Andrew; Liu, Christina; Stark, Azadeh; Chitale, Dhananjay; Greenlee, Robert; Multerer, Deborah; Honda, Stacey; Daida, Yihe; Feigelson, Heather Spencer; Glass, Andrew; Couch, Fergus J.; Rohan, Thomas E.; Ben-Dov, Iddo Z.

In: International Journal of Molecular Sciences, Vol. 18, No. 3, 627, 14.03.2017.

Research output: Contribution to journalArticle

Loudig, Olivier ; Wang, Tao ; Ye, Qian K. ; Lin, Juan ; Wang, Yihong ; Ramnauth, Andrew ; Liu, Christina ; Stark, Azadeh ; Chitale, Dhananjay ; Greenlee, Robert ; Multerer, Deborah ; Honda, Stacey ; Daida, Yihe ; Feigelson, Heather Spencer ; Glass, Andrew ; Couch, Fergus J. ; Rohan, Thomas E. ; Ben-Dov, Iddo Z. / Evaluation and adaptation of a laboratory-based cDNA library preparation protocol for retrospective sequencing of archived microRNAs from up to 35-year-old clinical FFPE specimens. In: International Journal of Molecular Sciences. 2017 ; Vol. 18, No. 3.
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AU - Lin, Juan

AU - Wang, Yihong

AU - Ramnauth, Andrew

AU - Liu, Christina

AU - Stark, Azadeh

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AU - Multerer, Deborah

AU - Honda, Stacey

AU - Daida, Yihe

AU - Feigelson, Heather Spencer

AU - Glass, Andrew

AU - Couch, Fergus J.

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AU - Ben-Dov, Iddo Z.

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N2 - Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.

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