Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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The effect of long-term -80°C storage of thyroid biospecimens on RNA quality and ensuring fitness for purpose.

Author(s): Mathieson W, Betsou F, Myshunina T, Pushkarev V, Pushkarev V, Shinkarkina A, Voskoboynyk L, Thomas GA

Publication: J Clin Pathol, 2016, Vol. 69, Page 1105-1108

PubMed ID: 27235537 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of frozen storage duration and tumor content on RIN using a total of 549 papillary thyroid carcinoma specimens of which 458 had a matched normal adjacent specimen. Specimens were collected during thyroidectomy over the span of 11 years (1998-2009). Within 30 min of removal, specimens were placed in 2 mL cryovials and snap-frozen by submersion in dry ice-cooled isopentane. Specimens were stored in -80˚C freezers in the country of collection and transported by an unspecified method to London. Specimens were homogenized using a TissueLyser and RNA extracted using the RNEasy mini kit. RNA was DNAse-treated and quantified by spectrophotometer. Integrity was evaluated by bioanalyzer.

   Summary of Findings:

   Storage duration was not correlated with RIN, but average RIN was higher in normal adjacent specimens than the matched tumor specimen (7.8 versus 7.6, P=0.004).

   Biospecimens

   • Tissue - Thyroid Gland

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Normal Adjacent
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Tumor Normal adjacent
   Storage	Storage duration	0.1-10.9 years

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2. Study Purpose

   This study investigated the effects of freeze-thaw cycling on RIN and compared maximum RNA amplicon size among frozen specimens of varying quality and formalin-fixed paraffin-embedded (FFPE) specimens. Sixty-four thyroidectomy specimens were snap-frozen by submersion in dry ice-cooled isopentane within 30 min of removal. During storage at -80˚C, forty specimens were thawed once while the remaining 26 were not thawed. Specimens were homogenized using a TissueLyser and RNA extracted using the RNEasy mini kit. RNA was extracted from an additional 12 FFPE specimens (no details of source or processing) using the RNEasy FFPE kit. RNA was DNAse-treated and quantified by spectrophotometer. Integrity was evaluated by bioanalyzer. Maximum amplicon size was determined by amplification of 65, 265, 534, and 942 bp fragments of HMBS.

   Summary of Findings:

   Of the 40 specimens subjected to a single freeze-thaw cycle, 39 had a RIN ≤2.8 and one had a RIN of 4.8, but 99% of the specimens that were not freeze-thaw cycled had a RIN >5. Similarly, the RNA from FFPE specimens had RIN <3. The 65 bp product was generated using RNA from all specimens including the FFPE specimens; however, amplification of the 265 bp and 534 bp products was possible in all frozen specimens, (regardless of RIN) but only 58% of FFPE specimens and none of the FFPE specimens, respectively.  In contrast, amplification of the 942 bp amplicon in frozen specimens was dependent on RIN with 100% success for specimens with a RIN >8.0 (frozen not thawed), 87% success for specimens with RIN between 5.0 and 8.0, and in none of the frozen specimens with a RIN <3.0 (freeze-thaw cycled).

   Biospecimens

   • Tissue - Thyroid Gland

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Normal Adjacent
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Snap frozen Formalin (buffered)
   RT-PCR Specific	Length of gene fragment	65 bp 265 bp 534 bp 942 bp
   RT-PCR Specific	Targeted nucleic acid	HMBS
   Storage	Freeze/thaw cycling	0 cycles 1 cycle

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