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

ABSTRACT: Unfixed tissue specimens most frequently are stored for long term research uses at either -80° C or in vapor phase liquid nitrogen (VPLN). There is little information concerning the effects such long term storage on tissue RNA or protein available for extraction. Aliquots of 49 specimens were stored for 5-12 years at -80° C or in VPLN. Twelve additional paired specimens were stored for 1 year under identical conditions. RNA was isolated from all tissues and assessed for RNA yield, total RNA integrity and mRNA integrity. Protein stability was analyzed by surface-enhanced or matrix-assisted laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS, MALDI-TOF-MS) and nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). RNA yield and total RNA integrity showed significantly better results for -80° C storage compared to VPLN storage; the transcripts that were preferentially degraded during VPLN storage were these involved in antigen presentation and processing. No consistent differences were found in the SELDI-TOF-MS, MALDI-TOF-MS or nLC-ESI-MS/MS analyses of specimens stored for more than 8 years at -80° C compared to those stored in VPLN. Long term storage of human research tissues at -80° C provides at least the same quality of RNA and protein as storage in VPLN.

SUBMITTER: Auer H

PROVIDER: S-EPMC4213858 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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

Auer H H Mobley J A JA Ayers L W LW Bowen J J Chuaqui R F RF Johnson L A LA Livolsi V A VA Lubensky I A IA McGarvey D D Monovich L C LC Moskaluk C A CA Rumpel C A CA Sexton K C KC Washington M K MK Wiles K R KR Grizzle W E WE Ramirez N C NC

Biotechnic & histochemistry : official publication of the Biological Stain Commission 20140506 7

Unfixed tissue specimens most frequently are stored for long term research uses at either -80° C or in vapor phase liquid nitrogen (VPLN). There is little information concerning the effects such long term storage on tissue RNA or protein available for extraction. Aliquots of 49 specimens were stored for 5-12 years at -80° C or in VPLN. Twelve additional paired specimens were stored for 1 year under identical conditions. RNA was isolated from all tissues and assessed for RNA yield, total RNA inte ...[more]

PMID: 24799092

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Project description:BACKGROUND: Interchange of genetically modified (GM) mice between laboratories using embryos provides several advantages. Not only is transport stress avoided, but also the health status of the recipient colony is not compromised. Embryos do not need to be shipped in frozen stage, which requires expensive packaging in addition to a certain degree of expertise in order to freeze and thaw them correctly. The aim of this study was to examine different storage conditions and their effect on embryo viability in order to establish the feasibility of practical, non-frozen conditions for embryo shipment. METHODS: Mouse morulae developed in vivo (collected from donors 2.5d post coitum) or in vitro (zygotes cultured until morulae stage) were stored, combining two different media (KSOMeq or KSOM-H) and temperatures (4 degrees C, 15 degrees C and 37 degrees C) throughout 24 or 48 hours. After storage in vitro viability was assessed determining percentage of development to blastocyst and total cell number. In vivo viability was determined based on the number of implantations and living fetuses after embryo transfer of stored embryos. The storage effect at the molecular level was assessed by studying a gene pool involved in early development by quantitative RT-PCR. RESULTS: In vivo-produced morulae stored for 24 hours did not show differences in development up to the blastocyst stage, regardless of the storage type. Even though a decrease in the total cell number in vivo was observed, embryo development after embryo transfer was not affected. All 24 hour storage conditions tested provided a similar number of implantations and fetuses at day 14 of pregnancy. Morulae obtained from in vitro embryo culture collected at the 1-cell stage showed a decreased ability to develop to blastocyst after 24 hours of storage at 15degrees C both in KSOMeq and KSOM-H. Concomitantly, a significant decrease of embryo implantation rates after transfer to recipients was also found. In order to further characterize the effect of non-frozen storage combining a molecular approach with the ordinary in vitro culture evaluation, embryos collected at the morula stage were submitted to the same storage conditions described throughout 48 hours. In vitro culture of those embryos showed a significant decrease in their developmental rate to blastocyst in both KSOMeq and KSOM-H at 15degrees C, which also affected the total number of cells. Gene transcription studies confirmed significant alterations in retrotransposons (Erv4 and Iap) after 48 h of storage at 15degrees C. CONCLUSIONS: Our results show that both KSOMeq and KSOM-H can be equally used, and that several temperature conditions allow good survival rates in vitro and in vivo. Some of these storage conditions can substitute freezing in order to maintain embryo viability for 24-48 hours, providing a reliable and less demanding technical alternative for embryo interchanges.

Project description:BackgroundReliable stability and yield of RNA from breast cancer tissues are important for biobanking, clinical trials, and diagnostic testing.MethodsAliquots of fresh primary tumor tissue from 17 surgically resected invasive breast cancers were placed into RNAlater at room temperature after tumor removal (baseline) and up to 3 hours thereafter or were snap frozen at baseline and 40 minutes thereafter. Samples were stored at -80°C until gene expression profiling with Affymetrix Human Gene U133A microarrays. We evaluated the effects of cold ischemic time (the time from tumor specimen removal to sample preservation) and sample preservation method on RNA yield, Bioanalyzer-based RNA integrity number, microarray-based 3'/5' expression ratios for assessing transcript integrity, and microarray-based measurement of single-gene and multigene expression signatures. The statistical significance of the effects was assessed using linear mixed effects regression models. All statistical tests were two-sided.ResultsSample preservation in RNAlater statistically significantly improved RNA integrity compared with snap freezing as assessed by the RNA integrity number, which increased from 7.31 to 8.13 units (difference = 0.82 units, 95% confidence interval [CI] = 0.53 to 1.11 units, P < .001), and RNA yield, which increased threefold from 8.9 to 28.6 μg (difference = 19.7 μg, 95% CI = 14.1 to 25.3 μg, P < .001). Prolonged cold ischemic delay at room temperature before sample stabilization decreased the RNA integrity number by 0.12 units/h (95% CI = 0.02 to 0.23 units/h) compared with a projected average RNA integrity number of 7.39 if no delays were present (P = .008) and decreased the RNA yield by 1.5 μg/h (95% CI = 0 to 4 μg/h) from a baseline mean RNA yield of 33.5 μg if no delays were present (P = .019). Prolonged cold ischemia statistically significantly increased the 3'/5' ratio of control gene transcripts, particularly of STAT1 (P < .001). Snap freezing statistically significantly increased the 3'/5' ratio of three control transcripts (ACTB, GAPDH, and 18S rRNA). Expression levels of single genes and multigene signatures for breast cancer were largely unaffected by sample preservation method or cold ischemia.ConclusionsSample preservation in RNAlater improves RNA yield and quality, whereas cold ischemia increases RNA fragmentation as measured by the 3'/5' expression ratio of control genes. However, expression levels of single genes and multigene signatures that are of diagnostic relevance in breast cancer were mostly unaffected by sample preservation method or prolonged cold ischemic duration.

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

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

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