Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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The Impact of Different Preservation Conditions and Freezing-Thawing Cycles on Quality of RNA, DNA, and Proteins in Cancer Tissue.

Author(s): Wang Y, Zheng H, Chen J, Zhong X, Wang Y, Wang Z, Wang Y

Publication: Biopreserv Biobank, 2015, Vol. 13, Page 335-47

PubMed ID: 26484573 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of different types of media and freeze-thaw cycles on RNA, DNA, and protein integrity in snap-frozen breast tissue. Specimens from 10 breast cancer patients undergoing surgery from May 2013 to Jan 2014 were divided into 0.2 x 0.2 x 0.2 cm3 aliquots at 4°C. Aliquots were then snap-frozen in liquid nitrogen in the absence of media, or in normal saline, Optimum Cutting Temperature compound (OCT), or following overnight preservation in RNAlater at 4°C. After 1 day, all specimens were then transferred from liquid nitrogen to -80°C, and subjected to 0, 1, 2, or 3 freeze-thaw cycles. Thawing consisted of specimen storage at room temperature for 1 h. Effects of freezing media and freeze-thaw cycling was investigated in 88 aliquots from 6 patients for RNA analysis and 60 aliquots from 5 patients for DNA and protein quality analysis. One aliquot from each specimen was H&E stained to ensure that tumor cell content was 70%. RNA was extracted using Trizol; analyzed with a bioanalyzer; and levels of nonmuscle cytoskeletal actins beta (ACTB), myelocytomatosis cellular oncogene (c-MYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), marker of proliferation Ki-67(MKI67), and proliferating cell nuclear antigen (PCNA) were determined by real-time RT-PCR. DNA was extracted using a TIANamp Genomic DNA Kit and analyzed by real-time RT-PCR for GAPDH, Cyclin-dependent kinase inhibitor 2A (P16), and Ras association domain-containing protein 1 (RASSF1a). Analysis of DNA methylation of eight CpGs in the first exon of RASSF1A (from CpG6 to CpG14) was performed by pyrosequencing. Proteins were extracted in RIPA Lysis Buffer, broken up ultrasonically, and expression of GAPDH and VEGFC proteins were examined by Western Blot. Band intensity was transformed into Integrated Optical Density (IOD) values for analysis.

   Summary of Findings:

   RNA integrity was comparable (RINs >9) among specimens snap-frozen in normal saline, OCT or in the absence of media, and those first preserved in RNAlater when specimens were not subjected to thawing. Differences among specimens frozen in different media were observed with freeze-thaw cycling. Specimens preserved in RNAlater before snap-freezing were least affected by freeze-thaw cycles with mean RINS of 9.4 ± 0.1, 9.1 ± 0.1, and 8.3 ± 0.3 after one, two, and three freeze-thaw events; respectively. Specimens snap-frozen in normal saline or OCT exhibited similar declines in mean RIN after one (7.3 ± 0.3 and 7.7 ± 0.3, respectively), two (6.6 ± 0.2 and 6.1 ± 0.3, respectively), and three freeze-thaw cycles (5.1 ± 0.6 and 5.5 ± 0.3, respectively). RIN in specimens snap-frozen in the absence of media were most affected by freeze-thaw cycling (6.7 ± 0.2 after one cycle, and 5.4 ± 0.4 after two cycles, and 4.0 ± 0.6 after three cycles). RINs were strongly and inversely correlated with real-time qRT-PCR Ct values for all five transcripts examined (GAPDH, PCNA, MYC, MKI67, and ACTB; r= -0.817, -0.762, -0.824, -0.781, and -0.807, respectively; P < 0.01 for all) and Ct values were relatively similar for specimens with RINs >5 but <8 when grouped by thaw time, but were lower for the RNAlater and OCT groups (data not provided). DNA amplification was unaffected by freezing media, as real-time qPCR Ct values did not differ significantly for all three genes (GAPDH, P16, and RASSF1a). Methylation of CpG islands of RASSF1a was significantly different between specimens snap-frozen in the absence of media and those frozen in OCT, normal saline, or preserved in RNAlater before snap-freezing for three patients (P< 0.05), while CpG island methylation was comparable among specimens snap-frozen in the absence of media and those frozen in OCT for the remaining two patients. While methylation levels were relatively similar among specimens snap-frozen in OCT or in the absence of media for all five patients, levels varied among specimens that were snap-frozen in normal saline or first preserved in RNAlater.  Protein integrity was also influenced by freezing media, as specimens snap-frozen in the absence of media had significantly higher IOD values for GAPDH than those that were snap-frozen in OCT or normal saline, or first preserved in RNAlater (p<0.05, all). Specimens snap-frozen in the absence of media also had higher VEGF-C IOD values compared to those snap-frozen in OCT (P<0.05), while those preserved in RNAlater before snap-freezing had the lowest IOD values.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Frozen
   • OCT
   • RNAlater

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR
   Protein	Western blot
   DNA	Bisulfite conversion assay
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Freeze/thaw cycling	1 cycle 2 cycles 3 cycles
   Biospecimen Preservation	Type of fixation/preservation	RNAlater Snap frozen OCT Saline
   Real-time qPCR Specific	Targeted nucleic acid	glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Cyclin-dependent kinase inhibitor 2A (P16) Ras association domain-containing protein 1 (RASSF1a)
   Western blot Specific	Targeted peptide/protein	GAPDH VEGFC
   Real-time qRT-PCR Specific	Targeted nucleic acid	non-muscle cytoskeletal actins beta (ACTB) myelocytomatosis cellular oncogene (c-MYC) marker of proliferation Ki-67(MKI67) proliferating cell nuclear antigen (PCNA) glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

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