Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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

Author(s): Choi Y, Kim A, Kim J, Lee J, Lee SY, Kim C

Publication: J Breast Cancer, 2017, Vol. 20, Page 393-399

PubMed ID: 29285045 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of formalin-fixation and storage duration of FFPE blocks on RNA quality, real-time RT-PCR amplification of short and long amplicons, and NGS results. Real-time PCR and NGS results were compared for FFPE specimens from two different centers. This study included a total of 143 FFPE and five frozen ER-positive breast cancer specimens, but details of specimen processing were not provided. The investigation of the effects of formalin-fixation was conducted using five paired frozen and FFPE specimens stored 1-2 years. The effects of storage duration, tumor size, and cellularity were investigated using fourteen specimens stored for 10 years (4 cases), 5 years (4 cases), and 1 year (6 cases). The performance of real-time PCR and NGS was investigated using 96 specimens collected at two different centers and stored 1-2 years. Cellularity was investigated by immunohistochemical staining of estrogen receptor. Tumor portions of 10 µm sections were deparaffinized for 20 min in an unspecified agent and digested with protease for 30 min to 16 h. RNA was isolated using the RecoverAll Total Nucleic Acid Isolation kit and quantified by spectrophotometry. RNA quality was assessed using the Agilent RNA 6000 Nano kit on a bioanalyzer.  mRNA was quantified by real-time PCR amplification of β-Actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and two different amplicons of ESR-1.  NGS libraries were prepared using the KAPA library preparation kit.

   Summary of Findings:

   RINS were low but comparable for matched FFPE and frozen specimens (1-2.4 versus 1.4-2.2). RNA yield and OD ratios were comparable among FFPE specimens archived for 1, 5, or 10 years, but RIN was modestly negatively correlated with storage duration (r=0.49, average of 1.5 for 10 years, 1.25 for 5 years, and 3.43 for 1 year). Real-time PCR CT values for ESR-1 and GAPDH were 2-3 cycles lower for 1 year-old specimens than for specimens stored for 10 years and were weakly to modestly correlated with storage duration (r=0.58 for ESR-1- 98 bp, r=0.38 for ESR-1-62 bp, and 0.39 for GAPDH). CT values were lower and the success rate higher for the shorter (62 bp) ESR-1 amplicon than the 98 bp ESR-1 amplicon (27.14-36.46 and 30.34-38.68 and 100% versus 71.4%, respectively). Further, the authors state that the shorter amplicon PCR was more efficient for the older FFPE specimens. Interestingly, FFPE blocks from the two different centers had different amplification success rates (78-88% and 41.3-47.8%) and average CT values were lower for specimens from the center with higher success rates (31.3-32.21 versus 36.02-36.91). Similarly, NGS was successful on all specimens from the center with 100% amplification success rate, but only 95.3% successful from specimens from the other center. Average FPKM for a given target differed greatly between centers.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing
   RNA	Real-time qRT-PCR
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Real-time qRT-PCR Specific	Targeted nucleic acid	GAPDH ESR-1 ACTB
   Real-time qRT-PCR Specific	Length of gene fragment	62 bp 98 bp
   Biospecimen Acquisition	Locale of biospecimen collection	Center A Center B

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