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The effects of frozen tissue storage conditions on the integrity of RNA and protein.

Author(s): Auer H, Mobley JA, Ayers LW, Bowen J, Chuaqui RF, Johnson LA, Livolsi VA, Lubensky IA, McGarvey D, Monovich LC, Moskaluk CA, Rumpel CA, Sexton KC, Washington MK, Wiles KR, Grizzle WE, Ramirez NC

Publication: Biotech Histochem, 2014, Vol. 89, Page 518-28

PubMed ID: 24799092 PubMed Review Paper? No

Purpose of Paper

The paper compared RNA yield, RNA integrity, mRNA integrity, and protein stability of normal and tumor tissue specimens stored at -80°C or the vapor phase of liquid nitrogen (VPLN) for 5 to 12 years.

Conclusion of Paper

Protein integrity and GAPDH and ACTB 3’/5’ ratios were comparable between tissue specimens stored at -80°C or in VPLN for prolonged periods of time, but total RNA yield and integrity were significantly higher for specimens stored at -80° C than those stored in VPLN.  Microarray and gene ontology analysis revealed higher signals in specimens stored at -80° C for 44 transcripts involved in antigen presentation and processing in comparison to levels observed with paired specimens stored in VPLN. Although data was not shown, the authors note that while a subset of specimens stored in VPLN were removed from the freezer more frequently than counterparts stored at -80° C, no correlation with RIN was observed.

Studies

  1. Study Purpose

    The study investigated the stability of RNA in biobanked tissue stored at - 140°C for 2 to 84 months using 189 fresh frozen head and neck tumor tissue specimens that contained a minimum of 80% malignant cells. Total RNA was isolated with TRIzol and quantified spectrophotometrically. RNA stability was quantified by RNA Integrity Number (RIN) using a bioanalyzer.

    Summary of Findings:

    Comparison of fresh frozen head and neck tumor tissue specimens stored at - 140°C for between 2 and 84 months showed no significant differences in mean RIN.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    RNA Spectrophotometry
    RNA Automated electrophoresis/Bioanalyzer
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration 2-8 months
    9-20 months
    21-41 months
    42-62 months
    63 – 84 months
    View more
  2. Study Purpose

    The study compared RNA yield, and RNA and mRNA integrity of various paired normal and tumor tissue specimens stored at -80°C or in vapor phase liquid nitrogen (VPLN). Sample sets included forty-nine paired remnant tumor tissue specimens were snap-frozen with or without OCT and stored at -80° C or in vapor phase liquid nitrogen (VPLN) for 5 -10 years.Tissue sections were hematoxylin and eosin-stained and examined by a pathologist for tumor content and histology. Total RNA was extracted using Tri Reagent, quantified spectrophotometrically, and analyzed with a bioanalyzer to determine RNA Integrity Number (RIN) and degradation factor, which has a scale of 1 to 100. RNA from 37 paired specimens with RIN > 1 and at least one of the specimens with a degradation factor < 100 were analyzed by microarray hybridization. mRNA integrity was assessed by 3’/5’ ratios of signal intensity of GAPDH and ACTB.

    Summary of Findings:

    RNA yield was statistically higher for specimens stored at -80° C than specimens stored in VPLN (mean = 7100 ng, SD 6800 ng vs. 4,400 ng, SD 5400 ng; P=0.006). Tissue specimens stored at -80°C also demonstrated superior integrity of total RNA as determined by RIN (5.8 vs. 4.3) and RNA degradation factor (39 vs. 56) compared to paired specimens stored in VPLN (P=0.0002 and P=0.003, respectively). However, GAPDH and ACTB 3’/5’ ratios, determined by microarray, were comparable among tissue specimens stored at -80°C or in VPLN (P=0.9 and P=0.5, respectively). Microarray analysis using 22,000 probe sets for over 18,000 transcripts revealed 44 transcripts with significantly higher signals in specimens stored at -80° C compared to those stored in VPLN, although the authors did not specify the magnitude of difference observed between storage temperatures. Gene ontology analysis of the 44 differentially expressed transcripts revealed many had roles associated with the biological processes of antigen presentation and processing, including endogenous and MHC class I antigens.

    Biospecimens
    Preservative Types
    • Frozen
    • OCT
    Diagnoses:
    • Neoplastic - Carcinoma
    • Neoplastic - Lymphoma
    • Neoplastic - Sarcoma
    • Neoplastic - Pediatric
    • Neoplastic - Normal Adjacent
    • Neoplastic - Benign
    Platform:
    AnalyteTechnology Platform
    RNA Spectrophotometry
    RNA Automated electrophoresis/Bioanalyzer
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration 5 – 12 years
    Storage Storage temperature -80°C
    vapor phase liquid nitrogen
    View more
  3. Study Purpose

    The study compared protein stability in various normal and tumor tissue specimens stored at -80°C or in the in vapor phase liquid nitrogen (VPLN).  Paired specimens from eight cases were snap-frozen with or without OCT and stored at -80° C or in VPLN for > 9 years. Specimens that were determined to contain equivalent tumor content and histology were analyzed by surfaced enhanced laser absorption/desorption time of flight mass spectrometry (SELDI-TOF-MS), matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), and nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/ MS).

    Summary of Findings:

    Protein analysis by SELDI-TOF-MS and MALDI-TOF-MS revealed qualitatively similar spectra for tissue specimens stored at -80°C or in VPLN. Spectra profiles were consistent within the two groups, as the coefficient of variation (CV%) was 0.92 for specimens stored at -80°C and 0.86 for specimens stored in VPLN. LC-ESI-MS/MS analysis revealed that 77% (183 of 239) of the unique peptides identified were common among specimens stored at different temperatures. The total number of proteins identified was also similar among specimens stored at -80°C and in VPLN (210 and 211, respectively), as was the number of independently identified proteins for each storage temperature (27 for -80°C and 29 for VPLN).

    Biospecimens
    Preservative Types
    • OCT
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    • Neoplastic - Pediatric
    • Neoplastic - Lymphoma
    • Neoplastic - Benign
    Platform:
    AnalyteTechnology Platform
    Protein MALDI-TOF MS
    Protein LC-ESI-MS/MS
    Protein SELDI-TOF MS
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration 9 – 12 years
    Storage Storage temperature -80°C
    vapor phase liquid nitrogen
    View more

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