Simplified and Versatile Method for Isolation of High-Quality RNA from Pancreas

Griffin, Michelle; Abu‐El‐Haija, Maisam; Haija, Marwa Abu El; Rokhlina, Tatiana; Uç, Aliye

doi:10.2144/0000113862

Cited by 25 publications

(15 citation statements)

References 12 publications

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“…It is possible that this material affected the efficiency of RNA extraction from these tissues, or, the lower yield may simply be due to lower overall cell density in samples with a large fraction of connective tissue. Using our methods, the tissue known to have the highest RNAse concentration—pancreas—did not produce RNA of inferior quality to any other tissue (Griffin et al 2012). If the data are presented for each individual case used (Table 4), the wide range of RIN values, including samples where no RIN value (N/A) could be obtained, indicates that there must be tissue-specific factors, not just perimortem or postmortem donor conditions, which affect RNA integrity.…”

Section: Resultsmentioning

confidence: 97%

Characterization of RNA isolated from eighteen different human tissues: results from a rapid human autopsy program

et al. 2016

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Many factors affect the integrity of messenger RNA from human autopsy tissues including postmortem interval (PMI) between death and tissue preservation and the pre-mortem agonal and disease states. In this communication, we describe RNA isolation and characterization of 389 samples from 18 different tissues from elderly donors who were participants in a rapid whole-body autopsy program located in Sun City, Arizona (www.brainandbodydonationprogram.org). Most tissues were collected within a PMI of 2–6 h (median 3.15 h; N = 455), but for this study, tissue from cases with longer PMIs (1.25–29.25 h) were included. RNA quality was assessed by RNA integrity number (RIN) and total yield (ng RNA/mg tissue). RIN correlated with PMI for heart (r = −0.531, p = 0.009) and liver (r = −558, p = 0.0017), while RNA yield correlated with PMI for colon (r = −485, p = 0.016) and skin (r = −0.460, p = 0.031). RNAs with the lowest integrity were from skin and cervix where 22.7 and 31.4 % of samples respectively failed to produce intact RNA; by contrast all samples from esophagus, lymph node, jejunum, lung, stomach, submandibular gland and kidney produced RNA with measurable RINs. Expression levels in heart RNA of 4 common housekeeping normalization genes showed significant correlations of Ct values with RIN, but only one gene, glyceraldehyde-3 phosphate dehydrogenase, showed a correlation of Ct with PMI. There were no correlations between RIN values obtained for liver, adrenal, cervix, esophagus and lymph node and those obtained from corresponding brain samples. We show that high quality RNA can be produced from most human autopsy tissues, though with significant differences between tissues and donors. The RNA stability and yield did not depend solely on PMI; other undetermined factors are involved, but these do not include the age of the donor.

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Section: Resultsmentioning

confidence: 97%

Characterization of RNA isolated from eighteen different human tissues: results from a rapid human autopsy program

et al. 2016

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“…When RIN values lower than 6.5 were found, in most cases this could be correlated with overall low cellular content or with notoriously low-yielding tissue types. 5 For some specific tissue types, such as pancreas and fatty (adipose) tissue, special RNA isolation procedures 14 and kits (Qiagen, RNeasy Lipid Tissue Mini Kit) are available. It may be useful to use these specialized kits in future tissue bank QC exercises, especially when a research group works with these tissue types.…”

Section: Discussionmentioning

confidence: 99%

Fit for Purpose Frozen Tissue Collections by RNA Integrity Number-Based Quality Control Assurance at the Erasmus MC Tissue Bank

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Arshad

et al. 2014

Biopreservation and Biobanking

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About 5000 frozen tissue samples are collected each year by the Erasmus Medical Center tissue bank. Two percent of these samples are randomly selected annually for RNA isolation and RNA Integrity Number (RIN) measurement. A similar quality assessment was conducted during centralization of a 20-year-old tissue collection from the cancer institute, a 15-year-old liver sample archive (-80°C), and a 13-year-old clinical pathology frozen biopsy archive (Liquid Nitrogen). Samples were divided into either high-quality (RIN ≥6.5) or low-quality overall categories, or into four "fit-for-purpose" quality groups: RIN <5: not reliable for demanding downstream analysis; 5 ≤RIN <6: suitable for RT-qPCR; 6 ≤RIN <8: suitable for gene array analysis; and RIN ≥8: suitable for all downstream techniques. In general, low RIN values were correlated with fatty, fibrous, pancreatic, or necrotic tissue. When the percentage of samples with RIN ≥6.5 is higher than 90%, the tissue bank performance is adequate. The annual 2011 quality control assessment showed that 90.3% (n=93) of all samples had acceptable RIN values; 97.4% (n=39) of the cancer institute collection had RIN values above 6.5; and 88.6% (n=123) of samples from the liver sample archive collection had RIN values higher than 6.5. As the clinical pathology biopsy collection contained only 58.8% (n=24) acceptable samples, the procurement protocols used for these samples needed immediate evaluation. When the distribution of RIN values of the different collections were compared, no significant differences were found, despite differences in average storage time and temperature. According to the principle of "fit-for-purpose" distribution, the vast majority of samples are considered good enough for most downstream techniques. In conclusion, an annual tissue bank quality control procedure provides useful information on tissue sample quality and sheds light on where and if improvements need to be made.

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“…The procedure to obtain high quality RNA varies between different laboratories [1]. The most reliable source for obtaining high quality RNA is by purification from freshly dissected tissues [2–7]. A factor to be taken into consideration when tissues are not processed immediately at the time of harvesting is the time samples are exposed to periods of warm ischemia, which may occur during and after surgery, and between sample thawing and processing.…”

Section: Introductionmentioning

confidence: 99%

“…The addition of an RNA stabilization solution to tissues, either by immersion or perfusion, has become standard practice for RNA stabilization [7, 10, 11]. Studies where select tissues are frozen immediately upon harvesting, with or without stabilization buffer, show no significant detriment to the quality of the RNA [6, 12–16].…”

Section: Introductionmentioning

confidence: 99%

Preservation of Multiple Mammalian Tissues to Maximize Science Return from Ground Based and Spaceflight Experiments

et al. 2016

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BackgroundEven with recent scientific advancements, challenges posed by limited resources and capabilities at the time of sample dissection continue to limit the collection of high quality tissues from experiments that can be conducted only infrequently and at high cost, such as in space. The resources and time it takes to harvest tissues post-euthanasia, and the methods and duration of long duration storage, potentially have negative impacts on sample quantity and quality, thereby limiting the scientific outcome that can be achieved.ObjectivesThe goals of this study were to optimize methods for both sample recovery and science return from rodent experiments, with possible relevance to both ground based and spaceflight studies. The first objective was to determine the impacts of tissue harvest time post-euthanasia, preservation methods, and storage duration, focusing on RNA quality and enzyme activities in liver and spleen as indices of sample quality. The second objective was to develop methods that will maximize science return by dissecting multiple tissues after long duration storage in situ at -80°C.MethodsTissues of C57Bl/6J mice were dissected and preserved at various time points post-euthanasia and stored at -80°C for up to 11 months. In some experiments, tissues were recovered from frozen carcasses which had been stored at -80°C up to 7 months. RNA quantity and quality was assessed by measuring RNA Integrity Number (RIN) values using an Agilent Bioanalyzer. Additionally, the quality of tissues was assessed by measuring activities of hepatic enzymes (catalase, glutathione reductase and GAPDH).ResultsFresh tissues were collected up to one hour post-euthanasia, and stored up to 11 months at -80°C, with minimal adverse effects on the RNA quality of either livers or RNAlater-preserved spleens. Liver enzyme activities were similar to those of positive controls, with no significant effect observed at any time point. Tissues dissected from frozen carcasses that had been stored for up to 7 months at -80°C had variable results, depending on the specific tissue analyzed. RNA quality of liver, heart, and kidneys were minimally affected after 6–7 months of storage at -80°C, whereas RNA degradation was evident in tissues such as small intestine, bone, and bone marrow when they were collected from the carcasses frozen for 2.5 months.ConclusionThese results demonstrate that 1) the protocols developed for spaceflight experiments with on-orbit dissections support the retrieval of high quality samples for RNA expression and some protein analyses, despite delayed preservation post-euthanasia or prolonged storage, and 2) many additional tissues for gene expression analysis can be obtained by dissection even following prolonged storage of the tissue in situ at -80°C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to spaceflight experiments that entail on-orbit sample recovery by astronauts.

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Simplified and Versatile Method for Isolation of High-Quality RNA from Pancreas

Cited by 25 publications

References 12 publications

Characterization of RNA isolated from eighteen different human tissues: results from a rapid human autopsy program

Characterization of RNA isolated from eighteen different human tissues: results from a rapid human autopsy program

Fit for Purpose Frozen Tissue Collections by RNA Integrity Number-Based Quality Control Assurance at the Erasmus MC Tissue Bank

Preservation of Multiple Mammalian Tissues to Maximize Science Return from Ground Based and Spaceflight Experiments

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