RNase H2-Dependent Polymerase Chain Reaction and Elimination of Confounders in Sample Collection, Storage, and Analysis Strengthen Evidence That microRNAs in Bovine Milk Are Bioavailable in Humans

Lanfang Wang; Mahrou Sadri; David Giraud; Janos Zempleni

doi:10.1093/jn/nxx024

. 2018 Jan 25;148(1):153–159. doi: 10.1093/jn/nxx024

RNase H2-Dependent Polymerase Chain Reaction and Elimination of Confounders in Sample Collection, Storage, and Analysis Strengthen Evidence That microRNAs in Bovine Milk Are Bioavailable in Humans

Lanfang Wang ^1,^b, Mahrou Sadri ¹, David Giraud ¹, Janos Zempleni ^1,^✉

PMCID: PMC6251634 PMID: 29378054

Abstract

Background

Evidence suggests that dietary microRNAs (miRs) are bioavailable and regulate gene expression across species boundaries. Concerns were raised that the detection of dietary miRs in plasma might have been due to sample contamination or lack of assay specificity.

Objectives: The objectives of this study were to assess potential confounders of plasma miR analysis and to detect miRs from bovine milk in human plasma.

Methods

Potential confounders of plasma miR analysis (circadian rhythm, sample collection and storage, calibration, and erythrocyte hemolysis) were assessed by quantitative reverse transcriptase polymerase chain reaction (PCR) by using blood from healthy adults (7 men, 6 women; aged 23–57 y). Bovine miRs were analyzed by RNase H2–dependent PCR (rhPCR) in plasma collected from a subcohort of 11 participants before and 6 h after consumption of 1.0 L of 1%-fat bovine milk.

Results

The use of heparin tubes for blood collection resulted in a complete loss of miRs. Circadian variations did not affect the concentrations of 8 select miRs. Erythrocyte hemolysis caused artifacts for some miRs if plasma absorbance at 414 nm was >0.300. The stability of plasma miRs depended greatly on the matrix in which the miRs were stored and whether the plasma was frozen before analysis. Purified miR-16, miR-200c, and cel-miR-39 were stable for ≤24 h at room temperature, whereas losses equaled ≤80% if plasma was frozen, thawed, and stored at room temperature for as little as 4 h. rhPCR distinguished between bovine and human miRs with small variations in the nucleotide sequence; plasma concentrations of Bos taurus (bta)-miR-21-5p and bta-miR-30a-5p were >100% higher 6 h after milk consumption than before milk consumption.

Conclusions

Confounders in plasma miR analysis include the use of heparin tubes, erythrocyte hemolysis, and storage of thawed plasma at room temperature. rhPCR is a useful tool to detect dietary miRs.

Keywords: confounders, microRNA, bovine milk, plasma, stability

Introduction

MicroRNAs (miRs) are ∼22 nucleotides long (1), hybridize with complementary sequences in the 3′-untranslated regions in mRNA (1), and silence genes through destabilizing mRNA or preventing translation of mRNA (2, 3). More than 60% of human protein-coding genes have been under selective pressure to maintain pairing to miRs, and 1881 high-confidence miRs are encoded in the human genome (4–6). It is safe to propose that miRs regulate virtually every gene network in humans and play roles in virtually all physiologic and pathological conditions in humans (7, 8).

miRs are encoded by their own genes, or by introns and exons of long non–protein-coding transcripts, and therefore can be obtained through endogenous synthesis (9–11). Endogenous synthesis of miRs is essential, and loss of miR maturation in Drosha and Dicer knockout mice is embryonically lethal (12–14). The essentiality of endogenous synthesis notwithstanding, evidence suggests that the dietary intake of miRs in plants and milk contributes to the body pool of miRs in humans and mice (15, 16). Strong momentum is building in support of the theory that foreign RNAs in micro-organisms, plants, and milk are bioavailable in humans and animals, particularly if RNAs are encapsulated in exosomes (17–34). For example, data from RNA sequencing analyses suggest that human transcripts make up only ≤60% of RNAs in human plasma (17). In addition, there is no reasonable doubt that small, interfering RNAs in genetically modified plants are absorbed by pests causing their death (35, 36). A considerable fraction of dietary (and endogenous) RNA is encapsulated in exosomes, which confers resistance to the harsh conditions in the gastrointestinal tract and facilitates the cellular uptake of miRs, including that in intestinal cells (23, 26, 33, 37, 38).

The first reports of biological activities of dietary miRs were met with appropriate skepticism due to concerns with regard to ineffective miR delivery and sample contamination (39–43). These discussions have slowed the rate of discovery in studies of the biological activity of dietary exosomes and their RNA cargos. In a recent opinion article, we recommended that, although the biological activity of dietary miRs warrants further exploration, the controversy surrounding this field is not a good reason to abandon research in closely related fields such as dietary exosomes and RNAs other than miRs (44).

Here we sought to advance the field of dietary miRs through identifying potential confounders in sample collection, storage, and analysis. We also used RNase H2–dependent PCR (rhPCR) to study the bioavailability of miRs in bovine milk in humans. rhPCR was originally developed to detect single nucleotide polymorphisms but also holds great potential to detect foreign miRs that differ from host miRs by a single nucleotide (45).

Methods

Participants

The participants in the individual experiments were sampled from a total pool of 13 apparently healthy adults (7 men, 6 women) aged 23–57 y. Exclusion criteria included lactose intolerance, diagnosis of cancer or inborn errors of metabolism, use of any medication, pregnancy, and smoking. Sampling times and challenges with 1%-fat bovine milk obtained from a grocery store and characteristics of the populations in individual experimental cohorts are described in Results. In milk-feeding studies, 11 healthy adults (6 men, 5 women) consumed 1.0 L milk, adjusted by total body water and with the use of a 75-kg man as reference (16). Participants were instructed to not consume dairy products for ≥24 h before the milk challenge. All protocols were approved by the University of Nebraska–Lincoln Institutional Review Board (protocol: 20170216771EP), and participants signed informed consent forms.

Blood collection

A trained phlebotomist collected blood through venipuncture with the use of EDTA-coated tubes. In one experiment, blood was collected in heparin tubes to determine whether the binding of exosomes to heparin causes a loss of miRs in samples (46). Plasma and RBCs were separated by centrifugation (524 × g for 25 min) at room temperature and analyzed immediately or stored at −80°C, depending on the purpose of a given experiment. RBCs were hemolyzed by mixing on a vortex and added back to plasma samples to assess the effects of sample hemolysis on miR concentrations in plasma. The degree of hemolysis was assessed by measuring the absorbance at 414 nm as a marker for hemoglobin (47). Select samples were subject to storage at room temperature with and without previous freezing to assess miR stability. RNA was isolated by using miRNeasy serum/plasma kits following the manufacturer's recommendations (Qiagen, Inc.). cel-miR-39 was added to plasma samples to assess the efficiency of RNA extraction, as recommended by the kit's manufacturer. Studies of circadian variation were conducted with the objective to identify possible candidates that might serve as housekeeping controls in the future.

PCR analyses

The nucleotide sequences of all miRs assayed in this study were obtained from miRBase, version 21.0 (5, 6). The majority of samples were analyzed by using qRT-PCR with the use of reagents, including PCR primers, from Exiqon, Inc. The efficiency of reverse transcription was assessed by using synthetic mature miR-21, miR-155, and miR-423 (Integrated DNA Technologies, Inc.). These miRs vary in their length and nucleotide composition and therefore capture features that might affect reverse transcription; their nucleotide sequences are identical in Homo sapiens (hsa) and Bos taurus (bta) (6). rhPCR was used to analyze miRs in a subset of plasma samples. rhPCR was originally developed to detect single nucleotide polymorphisms, which makes them powerful tools to discriminate endogenous and dietary miRs for which the nucleotide sequence may differ by as little as 1 nucleotide (45). rhPCR analyses were performed by using mature miR-21-5p and miR-30a-5p as models (hsa-miR-21-5p: 5′- UAGCUUAUCAGACUGAUGUUGA-3′; bta-miR-21-5p: 5′-UAGCUUAUCAGACUGAUGUUGACU-3′; hsa-miR-30a-5p: 5′-UGUAAACAUCCUCGACUGGAAG-3′; bta-miR-30a-5p: 5′-UGUAAACAUCCUCGACUGGAAGCU-3′) and synthetic miRs as authentic standards (Integrated DNA Technologies, Inc.) and the following GEN1-type species-specific forward PCR primers: bta-miR-21-5p = 5′-TAGCTTATCAGACTGATGTTGAcTAAAC-C3′ spacer phosphoramidite-3′; bta-miR-30a-5p = 5′-TGTAAACATCCTCGACTGGAAGcTAAAC-C3′ spacer phosphoramidite-3′ (Integrated DNA Technologies, Inc.). A proprietary universal reverse primer (Qiagen, Inc.) was used. Note that the nucleotide shown in lowercase is a ribonucleotide, as opposed to a deoxyribonucleotide. The 2^−△CT method was used to analyze qRT-PCR and rhPCR data (48). Under the conditions used in this study, we considered not detectable any miR that produced a cycle threshold (Ct) value of ≥29. For purposes of expressing miR levels in molar concentrations, PCR reactions were calibrated by using chemically synthesized authentic standards. Unless noted otherwise, the miR numbers used in this article refer to the human reference sequence in miRBase version 21 (5, 6). When nucleotide sequences of human and bovine miRs were identical, we omitted the identifiers hsa and bta.

Statistical analysis

Homogeneity of variances was confirmed by using Levene's test. Two-factor ANOVA and 1-factor ANOVA were used when testing the effects of 2 and 1 independent variables, respectively (49). For both the 2- and 1-factor ANOVAs, sample was included as blocking factor. For post hoc analyses, Dunnett's test was used when comparing treatments with a control, whereas Tukey's test was used when all possible comparisons were made. Calibration curves were generated by using linear regression analysis. Paired t test was used to assess the statistical significance of bta-miR-21-5p and bta-miR-30a-5p plasma concentrations before and after milk consumption. The R free software package version 3.3.3 was used for all statistical calculations (R Foundation). Data are reported as means ± SDs (means of triplicates from each participant). Effects of treatment were considered significant if P < 0.05.

Results

Assay calibration

The efficiency of reverse transcription was the same for the miRs that were tested. When synthetic miR-155, miR-21, and miR-423 were reverse transcribed and compared with synthetic DNA sequences, the signal produced by the RNA equaled 106.9% ± 8.6%, 104.0% ± 13.2%, and 104.8% ± 9.9%, respectively, to that of the synthetic DNA (P > 0.10 among miRs and compared with cDNA; n = 3). Calibration curves were created by using synthetic miR-21, miR-155, and miR-423 and were linear over the entire range of concentrations tested (R² = 1.00; Figure 1). The amplification efficiencies of the 3 miRs were >90% when using the theoretical maximum as denominator. We defined a Ct value of ∼29 as detection limit under the conditions used here.

Standard curves of synthetic miR-155 (A), miR-21 (B), and miR-423 (C) in qRT-PCR analysis; n = 3 independent samples for each data point and miR. Ct, cycle threshold; miR, microRNA.

Sampling procedures

The use of heparin tubes for blood collection resulted in a complete loss of plasma miRs. Blood from 3 healthy adults was collected in lithium heparin- and EDTA-coated tubes after an overnight fast. None of the 8 miRs tested (miR-103a, miR-16-5p, miR-423, miR-93, miR-191, miR-148b, miR-425, let-7i) were detectable (Ct values >31) if heparin was used as an anticoagulant. Ct values ranged from 19.0 ± 1.56 (miR-16-5p) to 25.9 ± 3.44 (let-7i) if EDTA was used as anticoagulant.

The degree of RBC hemolysis affected the apparent concentrations of some miRs in plasma. When samples with various degrees of hemolysis were created by adding hemolyzed RBCs to autologous plasma in samples from 3 healthy men, no change was observed for miR-23a and miR-451, whereas the apparent concentration of miR-16 increased significantly in plasma with an absorbance >0.300 at 414 nm (Figure 2). Note the increase in assay variability when working with RBC-enriched samples compared with other sets of samples in this study. All plasma samples used in subsequent experiments had an absorbance of ≤0.200 at 414 nm.

Effect of hemolysis on the concentrations of miR-16 (A), miR-451 (B) and miR-23a (C) in human plasma. Values are means ± SDs; n = 3 participants for each degree of hemolysis and miR, analyzed in triplicate. *P < 0.05 compared with absorbance of 0.150-0.160 at 414 nm. miR, microRNA.

No time-dependent variations in plasma miR concentrations were apparent during daytime. Blood was collected from 5 healthy adults (3 men, 2 women) at timed intervals in the morning and afternoon and analyzed for 8 miRs. The sampling time had no effect on plasma miR concentrations between 0830 and 1630 (Supplemental Figure 1). Among the miRs analyzed, miR-16, miR-423, and miR-191 appeared to be the least affected by circadian variations (see Discussion).

Stability

The stability of plasma miRs depended greatly on the matrix in which the miRs were stored and whether the plasma was frozen and thawed before further processing. If miRs were isolated from fresh human plasma (3 men) and stored at room temperature in RNase-free water for ≤24 h, degradation, if any, of miR-16, miR-200c, and cel-miR-39 compared with fresh plasma was not significant (P > 0.10; Figure 3). In contrast, if aliquots of the same plasma were kept at room temperature before RNA extraction, degradation of miRs was significant after 4–8 h of incubation and amounted to a loss of 60–80% of the 3 miRs at time 24 h compared with fresh plasma (Figure 4). If aliquots of the same plasma were frozen at −80°C and thawed before incubation at room temperature and RNA extraction, 40–70% of the 3 miRs were degraded after only 4 h of exposure to room temperature compared with fresh plasma; ∼80% of all 3 miRs were degraded after 24 h of exposure to room temperature compared with fresh plasma (Figure 5).

Stability of miR-29b (A), miR-200c (B), and miR-16 (C) in human plasma. Plasma was frozen overnight (–80°C) and kept at room temperature for ≤24 h before isolation of miRs. Time 0 h represents freshly isolated miRs. Values are means ± SDs; n = 3 participants for each time point and miR, analyzed in triplicate. *,**Compared with time 0 h: *P < 0.05 and **P < 0.01. miR, microRNA.

Stability of miR-29b (A), miR-200c (B), and miR-16 (C) in human plasma subjected to 1 freeze-thaw cycle. Plasma was frozen at –80°C overnight, thawed, and then kept at room temperature for ≤24 h before isolation of miRs. Time 0 h represents miRs isolated from plasma immediately after thawing. Values are means ± SDs; n = 3 participants for each time point and miR, analyzed in triplicate. *,**Compared with time 0 h: *P < 0.05 and **P < 0.01. miR, microRNA.

Bovine miRs

bta-miR-21-5p and bta-miR-30a-5p were detectable in human plasma by rhPCR after a milk meal. First, the rhPCR protocol was validated by using synthetic bta-miR-21-5p and hsa-miR-21-5p and an rhPCR primer specific for bta-miR-21-5p. If bta-miR-21-5p and hsa-miR-21-5p were amplified with primer for the bovine miR, Ct values were 16.7 ± 0.2 and 32.8 ± 0.3 (below the detection limit), respectively (P < 0.001; Supplemental Figure 2). Second, we analyzed plasma from the 11 individuals who participated in the milk-feeding study, sampled at time 0 h (before milk consumption) and 6 h after milk consumption by rhPCR and primers specific for bta-miR-21-5p and bta-miR-30a-5p. For bta-miR-30a-5p, Ct values were 29.7 ± 1.5 and 28.7 ± 1.8 at 0 and 6 h, respectively (P < 0.05). For bta-miR-21-5p, Ct values were 29.2 ± 3.6 and 27.0 ± 2.6 at 0 and 6 h, respectively (P < 0.05).

Discussion

This article advances studies of dietary RNAs at 2 important levels. First, a new technology was adopted and its suitability shown for detecting bovine miRs in human plasma, rhPCR. Second, we assessed and identified potential confounders of plasma miR analysis with the intent to develop a consensus of minimal experimental requirements in future studies of dietary miRs.

By using rhPCR, we detected an increase in bta-miR-21-5p and bta-miR-30a-5p in human plasma 6 h after milk consumption compared with before milk consumption. Given that the bioavailability of dietary miRs has proved to be a contentious issue, we would be surprised if this new information settled the discussion for once and all. We are currently completing a study that suggests that fluorophore-labeled RNAs, encapsulated in bovine-milk exosomes, are bioavailable in mice and accumulate in distinct tissues; that study also includes cross-fostering experiments that suggest that exosomes endogenously labeled with a fluorescent protein in porcine milk are absorbed by wild-type piglets nursed by a transgenic sow (50).

Two well-designed studies have raised doubts that the dietary transfer of miRs is quantitatively meaningful. Laubier et al. (41) used a transgenic mouse model that overexpresses miR-30b in mammary glands and secretes large amounts of miR-30b in milk to assess the bioavailability of miRs in pups. The transgenic mice secreted 134 times more miR-30b in milk than did wild-type mice. That ratio was diminished to an ∼31 times greater concentration of miR-30b in the stomach of pups nursed by transgenic dams compared with wild-type controls. We propose that the majority of miR-30b in the milk of transgenic mice was not encapsulated in exosomes and therefore both stability and bioavailability were compromised (23, 38). Laubier et al. (41) did not assess whether miR-30b was encapsulated in exosomes in murine milk.

Title et al. (42) also devised an elegant strategy to investigate the bioavailability of milk miRs and fostered miR-375 (and miR-200c) knockout pups to wild-type dams; they detected only trace amounts of miR-375 in plasma and peripheral tissues and concluded that absorption was minimal. We propose that miR-375 might actually have been absorbed and degraded after binding to its mRNA targets in the intestinal mucosa and liver before reaching the peripheral circulation and tissues. This is the classical concept of first-pass elimination (51). Note that artificial miR targets (“sponges”) have been used successfully to decrease the abundance of miRs in experimental settings (52). Title et al. (42) did not assess the expression of miR-375 targets in mucosa and liver of their mice, leaving room for interpretation. Interestingly, we did not observe a postprandial increase of plasma miR-375 in a human egg-feeding study, despite miR-375 being among the most abundant miR in eggs (D Fratantonio and J Zempleni, 2017).

This article suggests that the use of heparin tubes for blood collection, hemolysis of RBCs, and sample storage are quantitatively important confounders that may cause a near-complete loss of plasma miRs (heparin, storage) or an artificial increase of 300% (hemolysis). Our findings with regard to storage and stability are particularly noteworthy, because they cast some doubt on a previous report by Auerbach et al. (43), who proposed that miR-29b and miR-200c in bovine milk are not bioavailable in humans, contrary to a previous report (16). The dry ice used for shipping of the samples analyzed by Auerbach et al. (43) sublimated in transit and miRs were no longer detectable. Auerbach et al. showed that miR-16 and cel-miR-39, purified from plasma, were stable if kept at room temperature for ≤24 h. In this study we confirmed that purified miR-16 and cel-miR-39 (and miR-200c) are stable for ≤24 h, but we also showed that the vast majority of miRs were lost if plasma was frozen, thawed, and kept at room temperature. In light of these new observations the conclusions by Auerbach et al. can no longer be sustained.

The use of housekeeping miRs and internal standards is crucial in analyzing plasma miRs, whereas effects of circadian rhythm appear to be different for distinct species of miRs. For the 3 select miRs tested, we did not observe a difference with regard to the efficiency of reverse transcription. The use of internal standards is highly desirable because the percentage of recovery may be as little as 50% and varies greatly among the kits used for RNA extraction (53, 54). Note that for bioavailability studies of milk miRs it is not appropriate to use any RNA that is present in milk in high concentrations, because the RNA might be absorbed to an extent similar to that of the miRs under investigation. RNA sequencing data are available for bovine milk (55). The 5 miRs identified as candidates for housekeeping controls in this study were only studied in the context of circadian rhythm but not in milk-feeding studies or in severely stressed systems. Investigators are advised to assess whether the expression of the 5 putative housekeeping miRs changes in pathological conditions. In addition, some of the miRs that showed no circadian rhythm during daytime in this study showed time-dependent variations when samples were collected at night in a previous study (56).

We conclude that this study provides additional evidence in support of the theory that dietary miRs are bioavailable. The new evidence is based on the use of species-specific rhPCR and offers a straightforward explanation for why Auerbach et al. failed to detect bovine miRs in compromised human plasma samples (43, 45). Important next steps will include the identification of mechanisms by which the rather low concentrations of dietary (and nondietary) miRs change gene expression and the identification of phenotypes associated with depletion of dietary miRs.

Supplementary Material

Supplementary Data

Supplemental Figures 1 and 2 are available from the “Supplementary Data” link in the online posting of the article and from the same link in the online table of contents at https://academic.oup.com/jn/.

Click here for additional data file.^{(128.6KB, pdf)}

Acknowledgments

The authors’ responsibilities were as follows—LW and JZ: wrote the manuscript; LW, MS, and DG: conducted the research; JZ: had primary responsibility for final content; and all authors: analyzed the data, and read and approved the final manuscript.

Supported by the National Institute of Food and Agriculture (NIFA), USDA, under award 2015-67017-23181; NIH grant 1P20GM104320; NIFA 2016-67001-25301/NIH R01 DK107264; the Gerber Foundation; the Egg Nutrition Center; a Food for Health grant by the University of Nebraska President's Office; the University of Nebraska Agricultural Research Division (Hatch Act); and USDA multistate group W3002 (all to JZ).

Author disclosures: LW, MS, DG, and JZ, no conflicts of interest. The granting agencies had no influence on the study design; the collection, analysis, and interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication. JZ is a consultant for PureTech, Inc.

Abbreviations used

bta: Bos taurus
Ct: cycle threshold
hsa: Homo sapiens
miR: microRNA
rhPCR: RNase H2–dependent PCR

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35. Monsanto, Inc Petition for determination of nonregulated status for corn rootworm protected and glyphosate tolerant MON 87411 maize. St. Louis (MO): USDA Animal and Plant Health Inspection Service; 2013. Available from: https://www.aphis.usda.gov/brs/aphisdocs/13_29001p.pdf (accessed date April 1, 2017). [Google Scholar]
36. Heinemann JA, Agapito-Tenfen SZ, Carman JA. A comparative evaluation of the regulation of GM crops or products containing dsRNA and suggested improvements to risk assessments. Environ Int 2013;55:43–55. [DOI] [PubMed] [Google Scholar]
37. Izumi H, Kosaka N, Shimizu T, Sekine K, Ochiya T, Takase M. Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions. J Dairy Sci 2012;95:4831–41. [DOI] [PubMed] [Google Scholar]
38. Benmoussa A, Lee CH, Laffont B, Savard P, Laugier J, Boilard E, Gilbert C, Fliss I, Provost P. Commercial dairy cow milk microRNAs resist digestion under simulated gastrointestinal tract conditions. J Nutr 2016;146:2206–15. [DOI] [PubMed] [Google Scholar]
39. Snow JW, Hale AE, Isaacs SK, Baggish AL, Chan SY. Ineffective delivery of diet-derived microRNAs to recipient animal organisms. RNA Biol 2013;10:1107–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Dickinson B, Zhang Y, Petrick JS, Heck G, Ivashuta S, Marshall WS. Lack of detectable oral bioavailability of plant microRNAs after feeding in mice. Nat Biotechnol 2013;31:965–7. [DOI] [PubMed] [Google Scholar]
41. Laubier J, Castille J, Le Guillou S, Le Provost F. No effect of an elevated miR-30b level in mouse milk on its level in pup tissues. RNA Biol 2015;12:26–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Title AC, Denzler R, Stoffel M. Uptake and function studies of maternal milk-derived microRNAs. J Biol Chem 2015;290:23680–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Auerbach A, Vyas G, Li A, Halushka M, Witwer K. Uptake of dietary milk miRNAs by adult humans: a validation study. F1000Res 2016;5:721. [DOI] [PMC free article] [PubMed] [Google Scholar]
44. Zempleni J. Milk exosomes: beyond dietary microRNAs. Genes Nutr 2017;12:12. [DOI] [PMC free article] [PubMed] [Google Scholar]
45. Dobosy JR, Rose SD, Beltz KR, Rupp SM, Powers KM, Behlke MA, Walder JA. RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers. BMC Biotechnol 2011;11:80. [DOI] [PMC free article] [PubMed] [Google Scholar]
46. Balaj L, Atai NA, Chen W, Mu D, Tannous BA, Breakefield XO, Skog J, Maguire CA. Heparin affinity purification of extracellular vesicles. Sci Rep 2015;5:10266. [DOI] [PMC free article] [PubMed] [Google Scholar]
47. Kirschner MB, Kao SC, Edelman JJ, Armstrong NJ, Vallely MP, van Zandwijk N, Reid G. Haemolysis during sample preparation alters microRNA content of plasma. PLoS One 2011;6:1. [DOI] [PMC free article] [PubMed] [Google Scholar]
48. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402–8. [DOI] [PubMed] [Google Scholar]
49. Elston RC, Johnson WD. Essentials of biostatistics. Philadelphia: FA Davis Company; 1993. [Google Scholar]
50. Manca S, Giraud D, Zempleni J. The bioavailability and distribution of bovine milk exosomes is distinct from that of their cargos in mice [abstract]. FASEB J 2017;31:148.2.27677546 [Google Scholar]
51. Pond SM, Tozer TN. First-pass elimination: basic concepts and clinical consequences. Clin Pharmacokinet 1984;9:1–25. [DOI] [PubMed] [Google Scholar]
52. Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods 2007;4:721–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
53. Sanders I, Holdenrieder S, Walgenbach-Brunagel G, von Ruecker A, Kristiansen G, Muller SC, Ellinger J. Evaluation of reference genes for the analysis of serum miRNA in patients with prostate cancer, bladder cancer and renal cell carcinoma. Int J Urol 2012;19:1017–25. [DOI] [PubMed] [Google Scholar]
54. Khan N. Analysis of microRNAs in biological samples [dissertation]. Ottawa (Canada): University of Ottawa; 2015. [Google Scholar]
55. Sun J, Aswath K, Schroeder SG, Lippolis JD, Reinhardt TA, Sonstegard TS. MicroRNA expression profiles of bovine milk exosomes in response to Staphylococcus aureus infection. BMC Genomics 2015;16:806. [DOI] [PMC free article] [PubMed] [Google Scholar]
56. Heegaard NH, Carlsen AL, Lilje B, Ng KL, Ronne ME, Jorgensen HL, Sennels H, Fahrenkrug J. Diurnal variations of human circulating cell-free micro-RNA. PLoS One 2016;11. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib50] 50. Manca S, Giraud D, Zempleni J. The bioavailability and distribution of bovine milk exosomes is distinct from that of their cargos in mice [abstract]. FASEB J 2017;31:148.2.27677546 [Google Scholar]

[bib51] 51. Pond SM, Tozer TN. First-pass elimination: basic concepts and clinical consequences. Clin Pharmacokinet 1984;9:1–25. [DOI] [PubMed] [Google Scholar]

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[bib55] 55. Sun J, Aswath K, Schroeder SG, Lippolis JD, Reinhardt TA, Sonstegard TS. MicroRNA expression profiles of bovine milk exosomes in response to Staphylococcus aureus infection. BMC Genomics 2015;16:806. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib56] 56. Heegaard NH, Carlsen AL, Lilje B, Ng KL, Ronne ME, Jorgensen HL, Sennels H, Fahrenkrug J. Diurnal variations of human circulating cell-free micro-RNA. PLoS One 2016;11. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

RNase H2-Dependent Polymerase Chain Reaction and Elimination of Confounders in Sample Collection, Storage, and Analysis Strengthen Evidence That microRNAs in Bovine Milk Are Bioavailable in Humans

Lanfang Wang

Mahrou Sadri

David Giraud

Janos Zempleni