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.
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.
Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Olivier.loudig@einstein.yu.edu.
Olivier
O
Loudig
Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY 10461, USA. tao.wang@einstein.yu.edu.
Tao
T
Wang
Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY 10461, USA. kenny.ye@einstein.yu.edu.
Kenny
K
Ye
Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Juan.lin@einstein.yu.edu.
Juan
J
Lin
Department of Pathology, Rhode Island Hospital, Providence, RI 02903, USA. ywang6@Lifespan.org.
Yihong
Y
Wang
Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY 10461, USA. andrew.ramnauth12@myhunter.cuny.edu.
Andrew
A
Ramnauth
Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY 10461, USA. christina.liu@einstein.yu.edu.
Christina
C
Liu
Department of Pathology and Breast Oncology Program, Henry Ford Health System, Detroit, MI 48202, USA. ASTARK1@hfhs.org.
Azadeh
A
Stark
Department of Pathology and Breast Oncology Program, Henry Ford Health System, Detroit, MI 48202, USA. dchital1@hfhs.org.
Dhananjay
D
Chitale
Center for Clinical Epidemiology and Population Health, Marshfield Clinic Research Foundation, Marshfield, WI 54449, USA. greenlee.robert@mcrf.mfldclin.edu.
Robert
R
Greenlee
Center for Clinical Epidemiology and Population Health, Marshfield Clinic Research Foundation, Marshfield, WI 54449, USA. multerer.deborah@mcrf.mfldclin.edu.
Deborah
D
Multerer
Department of Pathology, Center for Health Research, Kaiser Permanente, 3288 Moanalua Road, Honolulu, HI 96819, USA. Stacey.Honda@kp.org.
Stacey
S
Honda
Department of Pathology, Center for Health Research, Kaiser Permanente, 3288 Moanalua Road, Honolulu, HI 96819, USA. Yihe.G.Daida@kp.org.
Yihe
Y
Daida
Center for Excellence in Cancer and Genomics, Kaiser Permanente Colorado, Denver, CO 80237, USA. heather.s.feigelson@kp.org.
Heather
H
Spencer Feigelson
Center for Health Research, Kaiser Permanente Northwest, Portland, OR 97227, USA. andy_5241@msn.com.
Andrew
A
Glass
Health Sciences Research, Mayo Clinic, Rochester, NY 55902, USA. couch.fergus@mayo.edu.
Fergus J
FJ
Couch
Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY 10461, USA. thomas.rohan@einstein.yu.edu.|Montefiore Medical Center, Bronx, NY 10467, USA. thomas.rohan@einstein.yu.edu.
Thomas
T
Rohan
Laboratory of Medical Transcriptomics, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel. Iddo@hadassah.org.il.
Iddo Z
IZ
Ben-Dov
D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D018270:Carcinoma, Ductal, Breast / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D005260:Female; D015723:Gene Library; D006801:Humans; D061986:MCF-7 Cells; D035683:MicroRNAs / Q000737:chemistry*; D016612:Paraffin Embedding / Q000379:methods* / Q000592:standards; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards
D035683:MicroRNAs
cDNA library preparation; ductal carcinoma in situ; formalin-fixed paraffin-embedded; invasive breast cancer; microRNAs; next-generation sequencing