回复“看来我们现在有证据和推理链条说明：转基因在理论 ...

随后，孟山都在Nature Biotechnology发文反驳之。孟山都通过实验得到了完全相反的结果，在权威杂志Nature Biotechnology上发表。

题目：Lack of detectable oral bioavailability of plant microRNAs after feeding in mice

作者：Brent Dickinson,Yuanji Zhang,Jay S Petrick,Gregory Heck,Sergey Ivashuta& William S Marshall

论文地址：Nature Biotechnology 31, 965–967 (2013) doi:10.1038/nbt.2737
http地址：http://www.nature.com/nbt/journal/v31/n11/full/nbt.2737.html

全文太长，只贴结论：
Overall, our results show neither apparent uptake of ingested plant miRNAs by mice nor regulation of target protein levels in liver and plasma or phenotypic changes in mice from ingested plant miRNAs that would be indicative of target gene regulation after rice feeding.



随后，孟山都在Nature Biotechnology发文反驳之。孟山都通过实验得到了完全相反的结果，在权威杂志Nature Biotechnology上发表。

题目：Lack of detectable oral bioavailability of plant microRNAs after feeding in mice

作者：Brent Dickinson,Yuanji Zhang,Jay S Petrick,Gregory Heck,Sergey Ivashuta& William S Marshall

论文地址：Nature Biotechnology 31, 965–967 (2013) doi:10.1038/nbt.2737
http地址：http://www.nature.com/nbt/journal/v31/n11/full/nbt.2737.html

全文太长，只贴结论：
Overall, our results show neither apparent uptake of ingested plant miRNAs by mice nor regulation of target protein levels in liver and plasma or phenotypic changes in mice from ingested plant miRNAs that would be indicative of target gene regulation after rice feeding.

张晨宇教授对此文的回复，并不同意孟山都的观点：
Reply to Lack of detectable oral bioavailability of plant microRNAs after feeding in mice
Xi Chen,         Ke Zen         & Chen-Yu Zhang
AffiliationsCorresponding authors
Nature Biotechnology 31, 967–969 (2013) doi:10.1038/nbt.2741

Xi Chen, Ke Zen and Chen-Yu Zhang reply:

We agree with Dickinson et al.1 that follow-up studies to carefully evaluate the uptake of food-derived microRNAs (miRNAs) and the potential of cross-kingdom gene regulation in animals are urgently required. However, we disagree with the conclusions of Dickinson et al.1 that nutritional differences in diet composition, rather than cross-kingdom gene regulation by plant miRNAs, are likely responsible for alterations in plasma low-density lipoprotein (LDL) following rice consumption. We feel that these authors fail to address several critical issues in detecting plant miRNAs in mouse plasma and liver samples.

In our study2, we first detected measurable plant miRNAs in the serum of human and mice, using deep sequencing. We then used oxidized deep sequencing to analyze miRNAs in the same sample set and demonstrated that plant miRNAs were successfully sequenced after oxidation, suggesting that they are genuine plant miRNAs containing 2′-O-methylated 3′-ends. However, when Dickinson et al.1 employed the Illumina (San Diego) Hiseq system to sequence small RNAs in rice-containing chow and rice grain, as well as in mouse liver and plasma samples after feeding mice with rice-containing chow and rice-based chow, no measurable uptake of any plant miRNAs was observed either in mouse liver or in plasma. In fact, they did not even detect enough plant miRNA signals in rice-containing chow or rice grain, a positive control for deep sequencing of rice miRNA.

After carefully analyzing the sequencing results by Dickinson et al.1, we note that their approach may have a sequencing bias between plant and animal miRNAs. Although endogenous mouse miRNAs were successfully measured in both plasma and liver, only 1,207 and 1,081 reads (per million raw reads) corresponded to rice miRNA detected in rice-containing chow and even rice grain. This is inconsistent with previous high-throughput sequencing studies in rice3, 4, 5, in which much more abundant rice miRNAs (generally 10% of total reads) have been detected. In addition, MIR156 and MIR168 are the most abundant miRNAs in rice grain and should account for at least 10,000 reads per million raw reads3, 4, 5, but the study by Dickinson et al.1 detects no MIR156 and only 192 and 153 reads, respectively, of MIR168 in rice-containing chow and rice grain. The extremely low levels of plant miRNAs suggests that the sequencing platform used in this study is insufficient to measure plant miRNAs. It is therefore not surprising that no rice miRNAs were detected in mouse liver and plasma.

In fact, their study highlights some critical issues in sequencing plant miRNAs in mammalian cells. A common step in high-throughput sequencing is the ligation of adaptor oligonucleotides using RNA ligase. Because plant miRNAs are 2′-O-methyl modified at the 3′ terminal nucleotide, and the 2′-O-methyl modification of small RNA 3′-ends can result in decreased ligation efficiency6, the sequencing procedure is biased against plant miRNAs compared with nonmodified animal miRNAs. In other words, if a sample is a mixture of 2′-O-methyl and 2′-OH small RNA, ligations would favor capture of the 2′-OH small RNAs. Thus, the convenient sequencing platform may result in low copy number of plant miRNAs in human and animal plasma. To efficiently capture small RNA from both animals and plants, one should optimize reaction parameters (e.g., extending the ligation time).

To further ensure that abundant mouse miRNAs do not have a negative impact on plant miRNAs during the sequencing procedure, Dickinson et al.1 spiked 10 ng of RNA isolated from rice grains into 1 mg of RNA isolated from mouse liver and used this mouse-plant RNA mix for small RNA library construction. However, plant RNA can never reach such a high level (1% of total RNA) in mouse liver. More importantly, analysis of sequencing results from the library prepared from rice grain RNA indicates again that the plant miRNAs are not sufficiently sequenced, as only ~10,000 reads per million reads of small RNAs were detected as plant miRNAs. For the mixed mouse-rice RNA library, the absolute sequencing reads instead of relative amounts should be shown. Without this information, it is difficult to evaluate whether the sequencing platform used in this study has the capability to detect plant miRNAs in mouse plasma and liver samples.

We would also like to draw readers' attention to the fact that even though Dickinson et al.1 mention that a survey of the animal small RNA data sets show no significant plant-derived miRNA accumulation in animal samples7, ours is not the only study to report small RNAs in animal plasma originating from ingested plant materials. Another study has reported that plasma contains exogenous small RNA sequences (e.g., MIR168) from common foods, including corn, rice, soybeans, tomato and grape8. Furthermore, a survey we have carried out of publicly available sequencing data reveals the presence of plant miRNAs in animal samples; for example, MIR156 and MIR168 have been detected in human peripheral blood mononuclear cell (GSM494809) and liver (GSM531978).

We note that there are several critical issues in measuring low levels of exogenous plant miRNAs in mammalian cells. The quantitative reverse trancriptase PCR (qRT-PCR) analysis conducted by Dickinson et al.1 of MIR168 in mouse plasma and liver after feeding mice with balanced rice chow and rice chow failed to detect uptake of MIR168 in mouse liver or plasma. We think several factors need to be carefully considered before drawing that conclusion.

First, the concentration of miRNAs should be within the linear working range of qRT-PCR. If the level of miRNA were too low, it would fall outside the working range of qRT-PCR and no difference would be detected. Thus, the absolute quantification method instead of relative quantification method should be employed to determine the levels of plant miRNAs in animal liver and plasma. It is also essential to include the experimental details about raw CT values, background signals and nonspecific amplification products, otherwise the robustness of the results is difficult to evaluate.

Second, an internal control or reference gene should be employed in the qRT-PCR assay to normalize miRNAs in plasma. Proper normalization is critical for quantitative analysis of extracellular miRNAs, as variations in the amount of material, sample collection, RNA extraction and enzymatic efficiency can introduce potential bias and contribute to quantification errors. In the study by Dickinson et al.1, synthetic Caenorhabditis elegans miRNAs (cel-miR-2 and cel-lin-4) were used as spike-in controls to normalize the levels of miRNAs in plasma. However, although most miRNAs are present in the plasma at the concentration of 1–1,000 fM, Dickinson et al.1 added 250 pmol of synthetic C. elegans miRNAs to 50 ml of plasma, making the final concentration too high to be used as an accurate internal control, which may even affect plant miRNA quantification in plasma. In our opinion, normalization of plant miRNAs to certain endogenous miRNAs is a better choice because endogenous miRNAs should not be substantially influenced by uptake of plant miRNAs.

To study the physiological relevance of plant-derived miRNAs in mammalian cells, our study also showed that MIR168 could regulate low-density lipoprotein receptor adaptor protein 1 (LDLRAP1) in human liver cells and influence the clearance of low density lipoprotein (LDL) from the blood. The elevation of plasma LDL-cholesterol levels could be largely reversed by MIR168 antisense oligonucleotide, confirming that the rice-mediated effect is specifically caused by MIR168. This conclusion is also supported by the observation that chow diet supplemented with mature MIR168 substantially enhanced the plasma LDL-cholesterol levels. Consistent with our finding, Dickinson et al.1 reported that animals eating rice had substantially increased plasma levels of LDL. However, they conclude that the increase in LDL levels they see results from nutritional imbalances between test and control groups rather than an RNA interference-mediated effect of consuming MIR168 in rice. We contend that more experiments are needed before the authors can exclude the contribution of MIR168. For example, some essential negative (mixing rice with MIR168 antisense oligonucleotide) or positive (mixing chow with mature MIR168) control should be considered in performing such experiments.

As further context for our work, we would also like to point readers to work by Eric Lam and co-workers at Rutgers University (F1000Prime doi:10.3410/f.13324007.14739062 (16 Nov 2011)). They have shown that rabbits take up siRNAs derived from transgenic tomatoes through the gastrointestinal tract; indeed, in April 2011, they filed a patent disclosure for the use of this technology to deliver small RNAs (including both siRNAs and miRNAs) for therapeutic purposes.

In summary, we would emphasize that understanding of the biological significance of the uptake of plant-derived miRNAs through food and the molecular mechanism by which plant miRNAs are absorbed and processed remains in its infancy. Future studies with improved experimental designs, careful controls and large sample size are required to validate and extend our findings.

张晨宇教授对此文的回复，并不同意孟山都的观点：
Reply to Lack of detectable oral bioavailability of plant microRNAs after feeding in mice
Xi Chen,         Ke Zen         & Chen-Yu Zhang
AffiliationsCorresponding authors
Nature Biotechnology 31, 967–969 (2013) doi:10.1038/nbt.2741

Xi Chen, Ke Zen and Chen-Yu Zhang reply:

We agree with Dickinson et al.1 that follow-up studies to carefully evaluate the uptake of food-derived microRNAs (miRNAs) and the potential of cross-kingdom gene regulation in animals are urgently required. However, we disagree with the conclusions of Dickinson et al.1 that nutritional differences in diet composition, rather than cross-kingdom gene regulation by plant miRNAs, are likely responsible for alterations in plasma low-density lipoprotein (LDL) following rice consumption. We feel that these authors fail to address several critical issues in detecting plant miRNAs in mouse plasma and liver samples.

In our study2, we first detected measurable plant miRNAs in the serum of human and mice, using deep sequencing. We then used oxidized deep sequencing to analyze miRNAs in the same sample set and demonstrated that plant miRNAs were successfully sequenced after oxidation, suggesting that they are genuine plant miRNAs containing 2′-O-methylated 3′-ends. However, when Dickinson et al.1 employed the Illumina (San Diego) Hiseq system to sequence small RNAs in rice-containing chow and rice grain, as well as in mouse liver and plasma samples after feeding mice with rice-containing chow and rice-based chow, no measurable uptake of any plant miRNAs was observed either in mouse liver or in plasma. In fact, they did not even detect enough plant miRNA signals in rice-containing chow or rice grain, a positive control for deep sequencing of rice miRNA.

After carefully analyzing the sequencing results by Dickinson et al.1, we note that their approach may have a sequencing bias between plant and animal miRNAs. Although endogenous mouse miRNAs were successfully measured in both plasma and liver, only 1,207 and 1,081 reads (per million raw reads) corresponded to rice miRNA detected in rice-containing chow and even rice grain. This is inconsistent with previous high-throughput sequencing studies in rice3, 4, 5, in which much more abundant rice miRNAs (generally 10% of total reads) have been detected. In addition, MIR156 and MIR168 are the most abundant miRNAs in rice grain and should account for at least 10,000 reads per million raw reads3, 4, 5, but the study by Dickinson et al.1 detects no MIR156 and only 192 and 153 reads, respectively, of MIR168 in rice-containing chow and rice grain. The extremely low levels of plant miRNAs suggests that the sequencing platform used in this study is insufficient to measure plant miRNAs. It is therefore not surprising that no rice miRNAs were detected in mouse liver and plasma.

In fact, their study highlights some critical issues in sequencing plant miRNAs in mammalian cells. A common step in high-throughput sequencing is the ligation of adaptor oligonucleotides using RNA ligase. Because plant miRNAs are 2′-O-methyl modified at the 3′ terminal nucleotide, and the 2′-O-methyl modification of small RNA 3′-ends can result in decreased ligation efficiency6, the sequencing procedure is biased against plant miRNAs compared with nonmodified animal miRNAs. In other words, if a sample is a mixture of 2′-O-methyl and 2′-OH small RNA, ligations would favor capture of the 2′-OH small RNAs. Thus, the convenient sequencing platform may result in low copy number of plant miRNAs in human and animal plasma. To efficiently capture small RNA from both animals and plants, one should optimize reaction parameters (e.g., extending the ligation time).

To further ensure that abundant mouse miRNAs do not have a negative impact on plant miRNAs during the sequencing procedure, Dickinson et al.1 spiked 10 ng of RNA isolated from rice grains into 1 mg of RNA isolated from mouse liver and used this mouse-plant RNA mix for small RNA library construction. However, plant RNA can never reach such a high level (1% of total RNA) in mouse liver. More importantly, analysis of sequencing results from the library prepared from rice grain RNA indicates again that the plant miRNAs are not sufficiently sequenced, as only ~10,000 reads per million reads of small RNAs were detected as plant miRNAs. For the mixed mouse-rice RNA library, the absolute sequencing reads instead of relative amounts should be shown. Without this information, it is difficult to evaluate whether the sequencing platform used in this study has the capability to detect plant miRNAs in mouse plasma and liver samples.

We would also like to draw readers' attention to the fact that even though Dickinson et al.1 mention that a survey of the animal small RNA data sets show no significant plant-derived miRNA accumulation in animal samples7, ours is not the only study to report small RNAs in animal plasma originating from ingested plant materials. Another study has reported that plasma contains exogenous small RNA sequences (e.g., MIR168) from common foods, including corn, rice, soybeans, tomato and grape8. Furthermore, a survey we have carried out of publicly available sequencing data reveals the presence of plant miRNAs in animal samples; for example, MIR156 and MIR168 have been detected in human peripheral blood mononuclear cell (GSM494809) and liver (GSM531978).

We note that there are several critical issues in measuring low levels of exogenous plant miRNAs in mammalian cells. The quantitative reverse trancriptase PCR (qRT-PCR) analysis conducted by Dickinson et al.1 of MIR168 in mouse plasma and liver after feeding mice with balanced rice chow and rice chow failed to detect uptake of MIR168 in mouse liver or plasma. We think several factors need to be carefully considered before drawing that conclusion.

First, the concentration of miRNAs should be within the linear working range of qRT-PCR. If the level of miRNA were too low, it would fall outside the working range of qRT-PCR and no difference would be detected. Thus, the absolute quantification method instead of relative quantification method should be employed to determine the levels of plant miRNAs in animal liver and plasma. It is also essential to include the experimental details about raw CT values, background signals and nonspecific amplification products, otherwise the robustness of the results is difficult to evaluate.

Second, an internal control or reference gene should be employed in the qRT-PCR assay to normalize miRNAs in plasma. Proper normalization is critical for quantitative analysis of extracellular miRNAs, as variations in the amount of material, sample collection, RNA extraction and enzymatic efficiency can introduce potential bias and contribute to quantification errors. In the study by Dickinson et al.1, synthetic Caenorhabditis elegans miRNAs (cel-miR-2 and cel-lin-4) were used as spike-in controls to normalize the levels of miRNAs in plasma. However, although most miRNAs are present in the plasma at the concentration of 1–1,000 fM, Dickinson et al.1 added 250 pmol of synthetic C. elegans miRNAs to 50 ml of plasma, making the final concentration too high to be used as an accurate internal control, which may even affect plant miRNA quantification in plasma. In our opinion, normalization of plant miRNAs to certain endogenous miRNAs is a better choice because endogenous miRNAs should not be substantially influenced by uptake of plant miRNAs.

To study the physiological relevance of plant-derived miRNAs in mammalian cells, our study also showed that MIR168 could regulate low-density lipoprotein receptor adaptor protein 1 (LDLRAP1) in human liver cells and influence the clearance of low density lipoprotein (LDL) from the blood. The elevation of plasma LDL-cholesterol levels could be largely reversed by MIR168 antisense oligonucleotide, confirming that the rice-mediated effect is specifically caused by MIR168. This conclusion is also supported by the observation that chow diet supplemented with mature MIR168 substantially enhanced the plasma LDL-cholesterol levels. Consistent with our finding, Dickinson et al.1 reported that animals eating rice had substantially increased plasma levels of LDL. However, they conclude that the increase in LDL levels they see results from nutritional imbalances between test and control groups rather than an RNA interference-mediated effect of consuming MIR168 in rice. We contend that more experiments are needed before the authors can exclude the contribution of MIR168. For example, some essential negative (mixing rice with MIR168 antisense oligonucleotide) or positive (mixing chow with mature MIR168) control should be considered in performing such experiments.

As further context for our work, we would also like to point readers to work by Eric Lam and co-workers at Rutgers University (F1000Prime doi:10.3410/f.13324007.14739062 (16 Nov 2011)). They have shown that rabbits take up siRNAs derived from transgenic tomatoes through the gastrointestinal tract; indeed, in April 2011, they filed a patent disclosure for the use of this technology to deliver small RNAs (including both siRNAs and miRNAs) for therapeutic purposes.

In summary, we would emphasize that understanding of the biological significance of the uptake of plant-derived miRNAs through food and the molecular mechanism by which plant miRNAs are absorbed and processed remains in its infancy. Future studies with improved experimental designs, careful controls and large sample size are required to validate and extend our findings.

张教授这篇论文只是学术研究，希望那些将其和转基因扯一起的童鞋，先把论文看懂了再说。不然，废话了一大篇，全都是扯淡。