The Stability of Medicinal Plant microRNAs in the Herb Preparation Process
Abstract
:1. Introduction
2. Results
2.1. Total RNA Isolation from Raw Herb Materials and Herb Preparations
2.2. The Stability of miRNA during the Herb Preparation Process
2.3. The Stability of Synthetic miRNAs
3. Discussion
4. Materials and Methods
4.1. Plant Materials
4.2. Preparations of Mistletoe Extracts
4.3. RNA Isolation
4.4. qRT-PCR
4.5. Treatment of Synthetic miRNAs
4.6. Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Sample Availability: Not available. |
RNA Parameters | Fresh Plant (FP) | Extract (FP), RT | Extract (FP)-Mechanical Treatment, RT | Dried Plant (DP) | Extract (DP)-Soaked, 80 °C | Extract (DP)-Boiled, 100 °C |
---|---|---|---|---|---|---|
RNA yield (µg/g) | 630.3 ± 73.5 | 1404.5 ± 531.2 | 1236.9 ± 482.8 | 398.2 ± 109.3 | 931.8 ± 212.7 | 26.9 ± 7.3 |
A260/A280 | 2.10 ± 0.03 | 2.04 ± 0.05 | 2.00 ± 0.07 | 2.02 ± 0.04 | 1.97 ± 0.03 | 1.67 ± 0.11 |
A260/A230 | 2.31 ± 0.16 | 2.44 ± 0.09 | 2.54 ± 0.09 | 2.30 ± 0.33 | 2.18 ± 0.15 | 0.80 ± 0.25 |
Plant miRNAs | Linear Range of Ct Values | Dynamic Quantification Range of miRNA Levels | Copy Number of miRNAs per Gram of Mistletoe Plants and Lyophilized Extracts | |||||
---|---|---|---|---|---|---|---|---|
Fresh Plant (FP) | Extract (FP), RT | Extract (FP)-Mechanical Treatment, RT | Dried Plant (DP) | Extract (DP)-Soaked, 80 °C | Extract (DP)-Boiled, 100 °C | |||
miR166a-3p | 13.1–28.5 | 1 nM–10 fM | 5.0 × 1011 ± 8.3 × 1010 | 7.4 × 109 ± 4.6×109 | 1.4 × 109 ± 3.0 × 108 | 1.2 × 1010 ± 6.1 × 109 | 4.2 × 108 ± 2.4 × 108 | NC |
miR159a | 17.8–32.9 | 1 nM–10 fM | 3.9 × 1011 ± 1.2 × 1011 | 1.1 × 1010 ± 5.3 × 109 | 3.4 × 109 ± 2.0 × 109 | 1.4 × 1010 ± 3.3 × 109 | 6.3 × 107 ± 3.0 × 107 | NC |
val-miR218 | 15.4–33.0 | 1 nM–10 fM | 7.9 × 109 ± 3.2 × 109 | 6.3 × 109 ± 3.3 × 109 | 2.0 × 109 ± 1.5 × 109 | 3.6 × 109 ± 1.8 × 109 | NC | NC |
val-miR11 | 20.7–32.6 | 100 pM–10 fM | 1.2 × 108 ± 6.4 × 107 | 4.2 × 107 ± 2.9 × 107 | NC | 2.4 × 107 ± 1.2 × 107 | NC | NC |
miR831-5p | 15.6–32.7 | 1 nM–1 fM | 3.3 × 107 ± 1.1 × 107 | 7.9 × 106 ± 3.3 × 106 | 2.9 × 106 ± 7.2 × 105 | 1.9 × 107 ± 6.1 × 106 | NC | NC |
has-let-7a | 20.9–32.7 | 100 pM–10 fM | NC | NC | NC | NC | NC | NC |
Synthetic miRNAs | Control | Mistletoe Extracts Containing Synthetic miRNAs | |||
---|---|---|---|---|---|
Extract (FP), RT | Extract (FP)-Mechanical Treatment, RT | Extract (DP)-Soaked, 80 °C | Extract (DP)-Boiled, 100 °C | ||
hsa-let-7a | 23.34 ± 0.47 | 34.81 ± 0.32 | 35.78 ± 0.79 | 35.63 ± 0.35 | 37.71 ± 0.58 |
2′-OMe hsa-let-7a | 23.08 ± 0.43 | 33.09 ± 0.24 | 35.30 ± 0.30 | 35.28 ± 0.70 | 37.14 ± 0.69 |
miRNAs | Sequence | RT Primer | Primer |
---|---|---|---|
miR166a-3p | UCGGACCAGGCUUCAUUCCCC | GTCGTATCCAGTGCGTGTCGTGGAGTCGGC AATTGCACTGGATACGACGGGGAA | F: AGTCGGACCAGGCTTCA R: CAGTGCGTGTCGTGGAG |
miR159a | UUUGGAUUGAAGGGAGCUCU | GTCGTATCCAGTGCGTGTCGTGGAGTCGGC AATTGCACTGGATACGACAGAGCT | F: GCCGTTTGGATTGAAGG R: CAGTGCGTGTCGTGGAG |
miR831-5p | AGGAAGACUGUAGAAGAGAUGAGG | GTCGTATCCAGTGCGTGTCGTGGAGTCGGC AATTGCACTGGATACGACCCTCAT | F: CTCAGGAAGACTGTAGAAGA R: CAGTGCGTGTCGTGGAG |
val-miR218 | GAUGAUCGCCACGUCGGAGGA | GTCGTATCCAGTGCAGGGTCCGAGGTATTC GCACTGGATACGACTCCTCC | F: AGGGATGATCGCCACG R: GTGCAGGGTCCGAGGT |
val-miR11 | CACUGUAGCACUUUUGACAAAG | GTCGT ATCCA GTGCA GGGTC CGAGG TATTC GCACT GGATA CGAC CTTTGT | F: GGGCACTGTAGCACTTTTG R: GTGCAGGGTCCGAGGT |
hsa-let-7a | UGAGGUAGUAGGUUGUAUAGUU | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTAT | F: GCCGTGAGGTAGTAGGTTGT R: GTGCAGGGTCCGAGGT |
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Xie, W.; Melzig, M.F. The Stability of Medicinal Plant microRNAs in the Herb Preparation Process. Molecules 2018, 23, 919. https://doi.org/10.3390/molecules23040919
Xie W, Melzig MF. The Stability of Medicinal Plant microRNAs in the Herb Preparation Process. Molecules. 2018; 23(4):919. https://doi.org/10.3390/molecules23040919
Chicago/Turabian StyleXie, Wenyan, and Matthias F. Melzig. 2018. "The Stability of Medicinal Plant microRNAs in the Herb Preparation Process" Molecules 23, no. 4: 919. https://doi.org/10.3390/molecules23040919