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Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues.

Author(s): Groelz D, Viertler C, Pabst D, Dettmann N, Zatloukal K

Publication: PLoS One, 2018, Vol. 13, Page e0203608

PubMed ID: 30192857 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to explore the potential effects of long-term FFPE and PFPE block storage duration and temperature on RNA yield, integrity, and suitability for qRT-PCR analysis. Tissue from eight different cases were used and included malignant liver and non-malignant stomach, liver, and duodenum tissues. Non-malignant and malignant specimens were not compared experimentally, nor were were the different tissue types. Each tissue specimen was aliquoted and snap-frozen in liquid nitrogen-cooled methyl butane or fixed in neutral buffered formalin or PAXgene Tissue Fixative for 3 (aliquots from 5 cases), 24 (aliquots from 7 cases), 72, 96, or 120 h (aliquots from 1 case each) at room temperature. Notably, the different fixation timepoints were designed to mirror the variability observed in clinical settings but fixation duration was not investigated experimentally. Fixed specimens were dehydrated through a grade series of ethanol then isopropanol, cleared in xylene, and impregnated in low-melting paraffin. FFPE and PFPE blocks were stored in the dark at ambient or 4°C. RNA was extracted from 10-20 (5 µm-thick) sections of FFPE blocks with Qiagen's RNeasy FFPE Kit, of PFPE blocks with PreAnalytiX's PAXgene Tissue RNA Kit, and from 20-70 mg of snap-frozen specimens with Invitrogen's TRIzol protocol. RNA concentration was determined by spectrophotometer and integrity was assessed by RNA integrity number (RIN) using a bioanalyzer. RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit and analyzed by qRT-PCR using Power SYBR Green PCR Master Mix for different sized amplicons of GAPDH (71, 153, 200, 277, 323 bp).

   Summary of Findings:

   RINs were higher among PFPE than FFPE tissue when stored for 1-2 months at either ambient (5.3 versus 3.4, respectively) or 4°C (5.6 versus 3.6), although statistical significance was not assessed. RINs declined rapidly within the first 6 months of block storage at ambient temperature for both FFPE and PFPE tissue and while 4°C storage slowed the rate, it did not reduce the magnitude of RIN decline. The authors note that the duration of fixation did not affect RIN among FFPE and PFPE tissue, but data was not shown.

   Cycle threshold (Ct) values from qRT-PCR analysis of GAPDH were dependent on amplicon length in all FFPE blocks with lower Ct values observed for the shortest amplicon (71 bp) compared to longer amplicons (153, 200, 277, 323 bp).  Effects of storage duration on qRT-PCR data was also influenced by the temperature of FFPE block storage with minimal differences (1-2 Ct values) observed among FFPE blocks stored for 1-72 months when stored at 4°C but robust differences (3-8 Ct values) observed when FFPE blocks were stored at ambient temperature. Similar trends in storage temperature and amplicon length were observed among PFPE blocks stored for 1-71 months at ambient temperature (change of 2-7 Ct values) while effects of storage duration and amplicon length were minimal (2 Ct values) among PFPE blocks stored at 4°C. When FFPE and PFPE archival blocks that were stored at ambient were compared to one another, delta Ct values were higher in FFPE than PFPE specimens for both short (71 bp) and medium (200 bp) length GAPDH amplicons. Delta Ct values for both FFPE and PFPE tissue increased with block storage at ambient temperature but remained stable when stored at 4°C. Delta Ct values were comparable among snap-frozen specimens and case-matched PFPE blocks stored at 4°C.

   Similarly designed experiments using a rat animal model mirrored the effects on RIN observed in human tissue specimens with the exceptions that RINs were higher (4.5-7) among blocks stored for the shortest duration (3 months) and investigation of additional storage temperatures revealed that FFPE and PFPE block storage at -20°C or -80°C prevented the decline altogether. Amplification of ACTB by qRT-PCR in rat tissue mirrored the trends observed in human specimens, although investigation of additional storage temperatures revealed superior qRT-PCR efficiency among FFPE and PFPE blocks stored at 4, -20, and -80°C compared to blocks stored at ambient temperature. Rat tissue PFPE blocks stored at -20 or -80°C had comparable ACTB Ct values to snap-frozen control specimens. DNA quality and suitability for qPCR analysis in rat tissues was more stable when FFPE and PFPE blocks were stored at 4 or -20°C for 9 y compared to those stored at ambient temperature and were superior among PFPE blocks when compared to FFPE blocks.

   Biospecimens

   • Tissue - Liver
   • Tissue - Small Bowel
   • Tissue - Stomach

   Preservative Types

   • Formalin
   • PAXgene

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Benign

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Targeted nucleic acid	GAPDH
   Real-time qRT-PCR Specific	Length of gene fragment	71 bp 153 bp 200 bp 277 bp 323 bp
   Storage	Storage temperature	Ambient 4°C
   Storage	Storage duration	1-2 months 3 months 6 months 12 months 24 months 36 months 60 months 72 months

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