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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.

Author(s): Loudig O, Wang T, Ye K, Lin J, Wang Y, Ramnauth A, Liu C, Stark A, Chitale D, Greenlee R, Multerer D, Honda S, Daida Y, Spencer Feigelson H, Glass A, Couch FJ, Rohan T, Ben-Dov IZ

Publication: Int J Mol Sci, 2017, Vol. 18, Page 627

PubMed ID: 28335433 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare RNA integrity and miRNA NGS expression profiles in matched frozen and FFPE specimens and to determine if the miRNA profiles  differed in DCIS patients who later developed invasive breast cancer and those who did not. The details of specimen procurement or FFPE processing were detailed. The effect of FFPE processing on RNA integrity and NGS profiles was investigated using matched frozen and FFPE specimens from a cervix (stored 8 years), a benign breast tumor (stored 4 years), and two invasive ductal carcinomas (stored 4-8 years). A larger clustering analysis of frozen and FFPE specimens was performed using the aforementioned specimens as well as three additional normal breast specimens stored for three years and three additional IBC cases stored for 1 year. To investigate if miRNA profiles in DCIS could indicate future progression to IBC, 22 DCIS specimens that later progressed to IBC and 22 that did not were selected based on matched patient age. Areas of interest were macrodissected on the day of extraction. RNA was extracted from frozen specimens after homogenization with a Tissue Tearor using Trizol. RNA was extracted from FFPE specimens by deparaffinization in Citri-Solv, a 1 h Proteinase K digestion at 59˚C, and a butanol-1 extraction followed by Trizol extraction. RNA was analyzed on a Bioanalyzer. Small-RNA sequencing was conducted using HiSeq 2500 Sequencing System after creation of a library by an optimized method. Expression differences in miR-221, miR-375, miR-363, and miR-20b were confirmed using TaqMan assays.

   Summary of Findings:

   As expected, electropherograms showed the 18s and 28s rRNA to be largely intact in frozen specimens but degraded in FFPE specimens, regardless of storage duration. The miRNA expression profiles of matched FFPE and frozen specimens clustered together with very strong correlations between them (r>0.93). Further, the two specimens from patients with invasive breast cancer clustered together. For the two cases of IBC, the correlations between matched specimens were higher than for the frozen specimens between cases. Plots of the ratios of miRNA for the two IBC cases in FFPE versus frozen specimens showed that FFPE specimens were able to differentiate between the cases. Clustering of the expression of the top 54 miRNA in three matched FFPE and frozen specimens and six additional FFPE specimens segregated specimens first by patient and then by diagnosis. Clustering based on the top 12 differentially expressed miRNA in DCIS created three groups one with 12 cases that progressed to invasive breast cancer within 6 months (cases), one group with 14 that did not progress to invasive breast cancer (controls) and one case that progressed, and a final group with nine cases that progressed and eight controls that did not. Six of the 12 miRNAs used were considered to have significantly higher expression in cases that progressed to invasive breast cancer as determined by NGS and real-time PCR.

   Biospecimens

   • Tissue - Breast
   • Tissue - Cervix

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Benign
   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Frozen Formalin (buffered)
   Preaquisition	Diagnosis/ patient condition	DCIS that progressed to IBC DCIS that did not progress IBC
   Storage	Storage duration	4 years 8 years

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2. Study Purpose

   The purpose of this study was to determine the minimum RNA input required for small-RNA library preparation from 35-year old FFPE benign breast specimen and 2 and 3-year old FFPE breast tumors. Areas of interest were macrodissected on the day of extraction. RNA was extracted from FFPE specimens by deparaffinization in Citri-Solv, a 1 h Proteinase K digestion at 59˚C, and a butanol-1 extraction followed by Trizol extraction. RNA was analyzed on a Bioanalyzer. Small-RNA sequencing was conducted using HiSeq 2500 Sequencing System after creation of a library by an optimized method. 

   Summary of Findings:

   Lower correlations among duplicates were observed when 50 ng of RNA was used compared to when 100 or 200 ng were used, but no differences between 100 ng and 200 ng were observed. The correlation in miRNA levels between libraries created 1 week apart from 100 ng of RNA from 9 archival specimens was >0.96. Importantly, the two tumor specimens clustered separately from the benign specimen, regardless of input amount.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Benign

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Benign Carcinoma
   Next generation sequencing Specific	Template/input amount	50 ng 100 ng 200 ng

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