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A Multivariate Evaluation of Factors Affecting the Quality of Freshly Frozen Tissue Specimens.

Author(s): Wang TH, Chen CC, Liang KH, Chen CY, Chuang WY, Ueng SH, Chu PH, Huang CG, Chen TC, Hsueh C

Publication: Biopreserv Biobank, 2017, Vol. 15, Page 344-349

PubMed ID: 28541714 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated effects associated with long-term storage of snap-frozen specimens at -80°C on RNA and DNA integrity. Differences between tumor and normal specimens and among different tissue types were also examined. A total of 1059 tumor and normal tissue specimens (21 brain, 44 head and neck, 136 liver, 72 breast, 88 renal, 142 gastric, 20 colorectal, and 104 lung) were collected and preserved between 2002 – 2011 following the same standard operating procedure. Each specimen was cut into 1 cm³ cubes, which were wrapped with aluminum foil, placed in cryogenic vials, immediately snap-frozen in liquid nitrogen, and then stored at -80°C. Genomic DNA was extracted from 627 specimens using a QIAmp DNA extraction kit and RNA was extracted from 496 specimens using an RNAeasy mini kit. All extractions were performed using a QIAcube automatic extraction machine. DNA integrity was assessed by successful PCR amplification of a fragment of GAPDH and RNA quality was assessed using a bioanalyzer and calculated RNA (RIN).

   Summary of Findings:

   RIN, a marker of RNA quality, declined significantly with frozen storage (P<0.0001). Mean RINs of specimens collected, snap-frozen, and stored beginning in 2002–2004, 2005–2008, and 2009–2011 groups were 5.04, 5.47, and 6.97, respectively. A total of 273 of the 496 (55%) specimens analyzed met the author-defined threshold for good quality RNA (RIN≥6). Applying this threshold, good quality RNA was isolated from 76.4% of specimens collected in 2009–2011, 50.5% of specimens collected in 2005–2008, and 44.6% of specimens collected in 2002–2004. Brain, breast, and stomach specimens yielded the lowest mean RINs (4.6, 5.62, and 5.88; respectively), and lower percentages of specimens that met the RIN threshold for good RNA quality (RIN≥6) (33.3%, 56.3%, and 55.3%, respectively; 50.7% combined) compared to other tissue types (69.1% combined). Comparably, the highest percentage of specimens that yielded RNA specimens of good quality were from lung, colorectal, and head and neck tumors (72.7%, 75%, and 75.6%; respectively). When compared, tumor specimens had significantly higher mean RINs than normal tissues (6.6 versus 5.6, P<0.0001) and yielded a larger percentage of specimens with RNA of good-quality (72.7% versus 49.7%, P<0.0001). Statistical analysis revealed higher percentages of low quality RNA among specimens collected and stored in 2002-2004 and 2005-2008 compared to those collected in 2009-2011 and stored for shorter durations (4.03- and 3.18-fold, respectively; P<0.001 for both), 2.17-fold higher rates for brain, breast, and stomach than other tissue types (P=0.0003), and 2.7-fold higher rates for normal specimens compared to tumor specimens (P<0.0001). Storage duration, tissue type, and cancer status were not associated with DNA quality for >97.5% of specimens in each group, demonstrated by successful amplification of an 800 bp fragment of GAPDH.

   Biospecimens

   • Tissue - Brain
   • Tissue - Breast
   • Tissue - Head and Neck
   • Tissue - Stomach
   • Tissue - Lung
   • Tissue - Colorectal
   • Tissue - Kidney
   • Tissue - Liver

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	brain breast liver kidney lung stomach colorectal
   PCR Specific	Targeted nucleic acid	GAPDH
   Storage	Storage duration	~ 5-15 years

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