Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk

Milk is a highly complex, heterogeneous biological fluid that contains non-nutritive, bioactive extracellular vesicles called exosomes. Characterization of milk-derived exosomes (MDEs) is challenging due to the lack of standardized methods that are currently being used for milk pre-processing, storage, and exosome isolation. In this study, we tested: 1) three pre-processing methods to remove cream, fat, cellular debris, and casein proteins from bovine milk to determine whether pre-processing of whole milk prior to long-term storage improves MDE isolations, 2) the suitability of two standard exosome isolation methods for MDE fractionation, and 3) four extraction protocols for obtaining high quality RNA from bovine and human MDEs. MDEs were characterized via Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western immunoblotting for CD9, CD63, and Calnexin protein markers. We also present an optimized method of TEM sample preparation for MDEs. Our results indicate that: 1) Removal of cream and fat globules from unpasteurized bovine milk, prior to long-term storage, improves the MDE yield but not purity, 2) Differential ultracentrifugation (DUC) combined with serial filtration is better suited for bovine MDE isolation compared to ExoQuick (EQ) combined with serial filtration, however both methods were comparable for human milk, and 3) TRIzol LS is better suited for RNA extraction from bovine MDEs isolated by EQ and DUC methods. 4) TRIzol LS, TRIzol+RNA Clean and Concentrator, and TRIzol LS+RNA Clean and Concentrator methods can be used for RNA extractions from human MDEs isolated by EQ, yet the TRIzol LS method is better suited for human MDEs isolated by DUC. The QIAzol + miRNeasy Mini Kit produced the lowest RNA yield for bovine and human MDEs.

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Comparison of methods for milk pre-processing, exosome isolation, and RNA extraction in bovine and human milk

10.1101/2020.08.14.251629 ◽

2020 ◽

Author(s):

Sanoji Wijenayake ◽

Shafinaz Eisha ◽

Zoya Tawhidi ◽

Michael A. Pitino ◽

Michael A. Steele ◽

...

Keyword(s):

Human Milk ◽

Biological Fluid ◽

Rna Extraction ◽

Bovine Milk ◽

Total Soluble Protein ◽

Long Term Storage ◽

Transmission Electron ◽

Whole Milk ◽

Membrane Bound

AbstractMilk is a highly complex, heterogeneous biological fluid that contains bioactive, membrane-bound extracellular vesicles called exosomes. Characterization of milk-derived exosomes (MDEs) is challenging due to the lack of standardized methods that are currently being used for milk pre-processing, exosome isolation, and RNA extraction. In this study, we tested: 1) three pre-processing methods to remove cream, fat, and casein proteins from bovine milk to determine whether pre-processing of whole milk, prior to long-term storage, improves MDE isolations, 2) two commonly-used exosome isolation methods, and 3) four extraction protocols for obtaining high quality MDE RNA from bovine and human milk. MDEs were characterized via Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). We also present an optimized method of TEM sample preparation and isolation of total soluble protein from MDEs. Our results indicated that: 1) pre-processing of bovine milk prior to storage does not affect the final exosome yield or the purity, 2) ExoQuick precipitation is better suited for MDE isolation than ultracentrifugation for bovine and human milk, and 3) TRIzol LS produced the highest RNA yield in bovine milk, whereas TRIzol LS, TRIzol+RNA Clean and Concentrator, and TRIzol LS+RNA Clean and Concentrator methods can be used for human milk.

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Dissolution Characteristics and Morphology of Large-sized Scorodite Particles Synthesized from Fe(II) and As(V) in Aqueous Solution

High Temperature Materials and Processes ◽

10.1515/htmp-2012-0080 ◽

2012 ◽

Vol 31 (4-5) ◽

pp. 451-458 ◽

Cited By ~ 3

Author(s):

S. Fujieda ◽

K. Shinoda ◽

T. Inanaga ◽

M. Abumiya ◽

S. Suzuki

Keyword(s):

Electron Microscopy ◽

Aqueous Solution ◽

Surface Defects ◽

Atmospheric Temperature ◽

Orthorhombic Structure ◽

X Ray ◽

Long Term Storage ◽

Transmission Electron ◽

Term Storage

AbstractA novel process for preparing scorodite particles with a diameter of approximately 20 µm from Fe(II) and As(V) in aqueous solution has been developed by DOWA Metals and Mining. In the present study, the dissolution characteristics of iron and arsenic from the scorodite particles synthesized by this process have been investigated under different conditions. The results show that the concentration of arsenic dissolved from the particles in aqueous solution is very low, but it has a complicated dependence on the temperature and pH of the solution. Transmission electron microscopy (TEM) with an energy dispersive X-ray spectrometer (EDS) was used to analyze the morphology, structure, and composition of the scorodite particles. The results indicate that the scorodite particles exhibit a nearly octahedral shape with planes composed of almost (111) planes in the orthorhombic structure. The concentration of iron at the surface of the particles is higher than that of iron inside of the particles. This characteristic morphology, along with the minimal surface defects of the scorodite particles, is considered to be responsible for the low dissolution of arsenic from the particles in aqueous solution. Atmospheric temperature and solution conditions were also found to be important for the safe, long-term storage of arsenic using scorodite particles.

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Improved long-term storage of hybridomas at −80° C using a bovine milk derivative

Journal of Immunological Methods ◽

10.1016/0022-1759(89)90072-0 ◽

1989 ◽

Vol 125 (1-2) ◽

pp. 13-18 ◽

Cited By ~ 4

Author(s):

F. Derouiche ◽

J.M. Bour ◽

C. Legrand ◽

J. Capiaumont ◽

F. Belleville ◽

...

Keyword(s):

Bovine Milk ◽

Long Term Storage ◽

Term Storage

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Evaluation of a low-cost procedure for sampling, long-term storage, and extraction of RNA from blood for qPCR analyses

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2014-1054 ◽

2015 ◽

Vol 53 (8) ◽

Cited By ~ 6

Author(s):

Rasmus B. Mærkedahl ◽

Hanne Frøkiær ◽

Lotte Lauritzen ◽

Stine B. Metzdorff

Keyword(s):

Gene Expression ◽

Clinical Trials ◽

Low Cost ◽

Rna Extraction ◽

Initial Step ◽

Rna Integrity ◽

Long Term Storage ◽

Significant Cost Reduction ◽

Term Storage

AbstractIn large clinical trials, where RNA cannot be extracted immediately after sampling, preserving RNA in whole blood is a crucial initial step in obtaining robust qPCR data. The current golden standard for RNA preservation is costly and designed for time-consuming column-based RNA-extraction. We investigated the use of lysis buffer for long-term storage of blood samples for qPCR analysis.Blood was collected from 13 healthy adults and diluted in MagMAX lysis/binding solution or PAXgene Blood RNA tubes and stored at –20 °C for 0, 1, or 4 months before RNA extraction by the matching method. RNA integrity, yield and purity were evaluated and the methods were compared by subsequent analyses of the gene expression levels ofThe MagMAX system extracted 2.3–2.8 times more RNA per mL blood, with better performance in terms of purity, and with comparable levels of integrity relative to the PAXgene system. Gene expression analysis using qPCR of: The MagMAX system can be used for storage of human blood for up to 4 months and is equivalent to the PAXgene system for RNA extraction. It furthermore, provides a means for significant cost reduction in large clinical trials.

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COMPARISON OF LONG-TERM STORAGE IN CHEMICAL FIXATIVES ON MORPHOLOGY AND ANATOMY OF SUPERDWARF WHEAT

HortScience ◽

10.21273/hortsci.31.6.918f ◽

1996 ◽

Vol 31 (6) ◽

pp. 918F-919

Author(s):

W.F. Campbell ◽

T. Strickland ◽

F.B. Salisbury ◽

P.S. Hole ◽

L. Gillespie ◽

...

Keyword(s):

Cellular Structure ◽

Vascular System ◽

Space Station ◽

Wheat Seedlings ◽

Long Term Storage ◽

Transmission Electron ◽

Electron Microscopical ◽

Cellular Components ◽

Term Storage

Wheat (Triticum aestivum L. cv. SuperDwarf) plants grown in the microgravity of space aboard the Russian space station Mir and sampled for morphological and anatomical comparison to those ex-posed to gravity on earth will be stored in chemical fixative for relatively long periods of time. Our objective was to examine, evaluate, and verify that the integrity of the vascular system and cellular components of wheat plants is maintained following storage in a chemical fixative. In ground-based studies, wheat seedlings were harvested on days 9, 28, and 68 and preserved in 4% formaldehyde: 1% glutaraldehyde (4F: 1G) fixative, adjusted to pH 7.2. Samples of leaves and/or stems were taken from these stocks after various times in storage (24 h, 1, 6, 12, and 24 months), dehydrated, and embedded in Spurr's resin. Semi-thin (1 μm) and thin (50–70 nm) sections were examined by light and transmission electron microscopy. Overall, there seems to be no major artifacts in the cellular structure. The plasmalemma and other organelles appeared normal in this fixative. Use of differential chromophores suggests that enzyme localizations at both the light and electron microscopical levels are not adversely affected by long-term storage. (Supported by NASA Grant NCC 2-831 and the Utah Agr. Expt. Station.)

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Tolerance of Stored Boar Spermatozoa to Autologous Seminal Plasma: A Proteomic and Lipidomic Approach

International Journal of Molecular Sciences ◽

10.3390/ijms21186474 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6474

Author(s):

Lisa Höfner ◽

Anne-Marie Luther ◽

Alessandra Palladini ◽

Thomas Fröhlich ◽

Dagmar Waberski

Keyword(s):

Mass Spectrometry ◽

Seminal Plasma ◽

Semen Analysis ◽

Computer Assisted ◽

Protein Markers ◽

Long Term Storage ◽

Lipidome Analysis ◽

Boar Spermatozoa ◽

Term Storage

Long-term exposure of liquid preserved boar spermatozoa to seminal plasma (SP) can cause dramatic sperm injury. This study examined whether boar specificity exists in the sensitivity of spermatozoa to SP and whether correspondent biomarkers can be identified. Consecutive ejaculates (n = 4–5) collected from 19 boars were centrifuged, diluted with a pH-stablising extender with 10% (v/v) autologous SP and evaluated by computer-assisted semen analysis and flow cytometry. Up until 144 h storage, four boars showed consistently high sperm motility, viability and mitochondria activity, and one boar showed consistently low values. Intra-boar variability was high in the other boars. Screening of SP (n = 12 samples) for protein markers using mass spectrometry identified three protein candidates of which the granulin precursor, legumain and AWN were 0.5 to 0.9 log2-fold less abundant (p < 0.05) in SP-resistant compared to SP-sensitive samples. Lipidome analysis by mass spectrometry revealed 568 lipids showing no difference between the SP-groups. The most abundant lipids were cholesterol (42,442 pmol), followed by phosphatidylserine (20,956 pmol) and ether-linked phosphatidylethanolamine (13,039 pmol). In conclusion, three candidate proteins were identified which might be indicative of SP-tolerance of sperm during long-term storage. Noteworthy, a first lipidomic profile of boar SP is presented.

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Combination of High-Pressure Processing and Freeze-Drying as the Most Effective Techniques in Maintaining Biological Values and Microbiological Safety of Donor Milk

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18042147 ◽

2021 ◽

Vol 18 (4) ◽

pp. 2147

Author(s):

Sylwia Jarzynka ◽

Kamila Strom ◽

Olga Barbarska ◽

Emilia Pawlikowska ◽

Anna Minkiewicz-Zochniak ◽

...

Keyword(s):

High Pressure ◽

Human Milk ◽

Nutritional Value ◽

Freeze Drying ◽

High Pressure Processing ◽

Microbiological Safety ◽

Long Term Storage ◽

Milk Banks ◽

Term Storage

Background: Human milk banks have a pivotal role in provide optimal food for those infants who are not fully breastfeed, by allowing human milk from donors to be collected, processed and appropriately distributed. Donor human milk (DHM) is usually preserved by Holder pasteurization, considered to be the gold standard to ensure the microbiology safety and nutritional value of milk. However, as stated by the European Milk Banking Association (EMBA) there is a need to implement the improvement of the operating procedure of human milk banks including preserving and storing techniques. Aim: The purpose of this study was to assess the effectiveness and safety of the selected new combination of methods for preserving donor human milk in comparison with thermal treatment (Holder pasteurization). Methods: We assessed (1) the concentration of bioactive components (insulin, adiponectin, leptin, activity of pancreatic lipase, and hepatocyte growth factor) and (2) microbiological safety in raw and pasteurized, high-pressure processed and lyophilization human breast milk. Results: The combination of two techniques, high-pressure processing and freeze-drying, showed the best potential for preserving the nutritional value of human milk and were evaluated for microbiological safety. Microbiological safety assessment excluded the possibility of using freeze-drying alone for human milk sample preservation. However, it can be used as a method for long-term storage of milk samples, which have previously been preserved via other processes. Conclusion: The results show that high-pressure treatment is the best method for preservation that ensures microbiological safety and biological activity but subsequent freeze-drying allowed long-term storage without loss of properties.

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Lipophagy contributes to long-term storage of spermatozoa in the epididymis of the Chinese soft-shelled turtle Pelodiscus sinensis

Reproduction Fertility and Development ◽

10.1071/rd18307 ◽

2019 ◽

Vol 31 (4) ◽

pp. 774 ◽

Cited By ~ 5

Author(s):

Hong Chen ◽

Yufei Huang ◽

Ping Yang ◽

Tengfei Liu ◽

Nisar Ahmed ◽

...

Keyword(s):

Molecular Mechanisms ◽

Sperm Storage ◽

Pelodiscus Sinensis ◽

Long Term Storage ◽

Transmission Electron ◽

Epididymal Spermatozoa ◽

Oil Red O Staining ◽

Term Storage ◽

Oil Red O

Spermatozoa are known to be stored in the epididymis of the Chinese soft-shelled turtle Pelodiscus sinensis for long periods after spermiation from the testes, but the molecular mechanisms underlying this storage are largely unknown. In this study, epididymal spermatozoa were investigated to determine the potential molecular mechanism for long-term sperm storage in P. sinensis. Transmission electron microscopy (TEM) and Oil red O staining indicated that unusually large cytoplasmic droplets containing lipid droplets (LDs) were attached to the epididymal spermatozoa. However, the content of LDs decreased gradually with the sperm storage. LDs were surrounded by autophagic vesicles and sequestered as degradative cargo within autophagosome. Immunofluorescence and western blotting demonstrated that autophagy in spermatozoa increased gradually with the storage time. Invitro studies found that spermatozoa obtained from soft-shelled turtles in January can survive more than 40 days at 4°C. Furthermore, immunofluorescence and TEM showed that autophagy was involved in the degradation of LDs with the extension of sperm incubation. Inhibition of autophagy with 3-methyladenine significantly suppressed LD degradation. Moreover, adipose triglyceride lipase was involved in the metabolism of LDs. These findings indicate that lipophagy was activated to maximise LD breakdown, which contributes to long-term sperm storage in the epididymis of P. sinensis.

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