Key Results
We achieved success in several parallel streams of research throughout this project:
- Functioning lyophilised discs were successfully synthesised for sandwich hybridisation detection.
- Extraction methods successfully yielded ath-miR399f from A. thaliana - this included an accessible RNA extraction protocol that farmers could use.
- Rolling Circle Amplification, in response to various ath-miR399f concentrations, was successfully visualised using a fluorescent ThT readout.
Functioning lyophilised discs were validated for hybridisation
Figure 1. Fluorescence of stored discs. This shows the differences in fluorescent intensity between stored discs with different binding methods and different storage techniques. The fluorescence of discs treated with ath-miR399f, and the two controls, hsa-miR16-5p and no miRNA, are shown. Error bars with +/- SD are given.
It was shown that ath-miR399f was able to be detected by lyophilised and fridge-stored discs. It showed a significantly higher fluorescence intensity than treated discs that had hsa-miR16-5p applied, indicating a specific detection of ath-miR399f. Prior experiments were performed in addition to this final experiment to validate the protocol at every step of the way with these being shown below.
Extraction methods successfully yielded ath-miR399f from A. thaliana
RT-qPCR performed on extracts obtained from each extraction method showed amplification of ath-miR399f, supported by 1) Cq values, 2) amplification plots and 3) gel electrophoresis of qPCR products.
The Cq values obtained for the different extraction methods, when the primers we designed were used, all differed significantly to the no-template control (NTC), according to a post-hoc Tukey’s Honest Significant Difference test (p < 0.05) (Figure 2). Moreover, these wells were assigned the “AMP” status, reinforcing the view that amplification occurred in these wells, unlike their no reverse transcriptase counterparts, which were all flagged as “NO_AMP”. A similar case was observed when primers from Thermo Fisher Scientific’s TaqMan™ miRNA Assay were used, though the Cq values obtained here were substantially lower, with values between 24 and 26.
Figure 2. ath-miR399f detected from different extraction methods, using different primers. Cq were values obtained from each detection method, when DIY and TF primers were used, separately. Undetermined Cq values were obtained for some no reverse transcriptase (RT) controls, so no bars were plotted for these wells. WHT: Whatman, ISP: Isopropanol, FRM: Farmer extraction, TRZ: TRIzol extraction.
Amplification plots derived from each condition supported the conclusion that amplification was occurring, where exponential and plateau phases could be observed (Figure 3). Moreover, these plots were visibly distinct from the no reverse transcription controls, which were largely flagged as “NO_AMP” or occasionally, “Inconclusive”.
Figure 3. Amplification plots for each extraction method, corresponding no reverse transcription controls, and NTC, detected using A) primers designed according to literature and B) primers from Thermo Fisher Scientific’s TaqMan™ miRNA Assay, against ath-miR399f.
Lastly, gel electrophoresis also provided evidence that a single product was being amplified when primers from Thermo Fisher Scientific’s TaqMan™ miRNA Assay were used, indicating that ath-miR399f was being extracted using the different extraction methods (Figure 4).
Figure 4. Agarose gel electrophoresis of qPCR products from the different extraction methods, where bands are present for the 5 different extraction methods, detected using primers from Thermo Fisher Scientific’s TaqMan™ miRNA Assay. Two ladder compositions were also tested - Ladder (dilute): 6-fold diluted ladder, Ladder (normal): 3-fold diluted ladder. ‘8’: 3 x 108 copies of miRNA, ‘4’: 3 x 104 copies of miRNA, ntc: no-template control. ath-miR399f: using primers against ath-miR399f, hsa-miR16-5p: using primers against hsa-miR16-5p.
For more information regarding the design and development of this assay, see RT-qPCR results.
Solution-phase RCA-G-quadruplex shown to be quantitative
To investigate the dependence of the fluorescent signal on the concentration of ath-miR399f, we varied the concentration of ath-miR399f from 75 nM to 7.5 pM and tested with our G-quadruplex assay. We also examined the selectivity of the assay by supplementing miR16, which, as mentioned before, is 56% identical to ath-miR399f. In addition, we included a control without Thioflavin T (ThT) to assess the intrinsic fluorescence of the RCA product and components. As illustrated in Figure, the signal varies with the concentration of ath-miR399f - the higher the ath-miR399f concentration, the more intense the fluorescence signal. The emission peak corresponded approximately to 486 nm as supported in another work1 which collected their emission at 490 nm. We have also addressed the plateau-shaped graph from the first measurement by adjusting the gain down from 90% to 20%. The graphs confirmed that without ThT, there was a substantially lower fluorescence readout. With regards to selectivity, at the same concentration, ath-miR399f produced a signal at 486 nm that was around 11 times that of miR16, which could be concluded that there was some selectivity for ath-miR399f to which the probe is designed to bind. Compared to the negative control, the signal emitted from the condition with 75 nM (effective concentration 2.5 nM) ath-miR399f was around 38-fold that of the negative control.
Figure 5. Shows how fluorescence signal varies with the concentration of ath-miR399f when Thioflavin T (ThT) is added, the intrinsic fluorescence activity of an RCA product from ath-miR399f without ThT and the assay’s selectivity with an RCA reaction performed with hsa-miR16-5p and ThT. Spectral scans support the concentration dependence of the signal on the ath-miR399f concentration. Lower fluorescence readout observed in the absence of ThT, highlighting ThT as the fluorescence output source. Spectral scan of condition with hsa-miR16-5p (75 nM) comparable to that of condition with ath-miR399f at 100-fold dilution (750 pM).
The fluorescent readout could, furthermore, be used to plot a standard curve. A semi-log standard curve was plotted for fluorescence and the logarithmic scale of ath-miR399f concentration with nanomolar (nM) units as shown in Figure. We excluded the data points with a negative fluorescence relative to blank. The detection limit - 27 nM or effective concentration of 900 pM due to the dilution from adding other components - was calculated from this standard plot with the formula that detection limit equals to 3*Standard deviation of Regression divided by Slope of the curve2.
Figure 6. Semi-log Standard curve of Solution-phase G-quadruplex ThT assay (20% Gain). Shows how the fluorescence activity of RCA products from ath-miR399f G-quadruplex circular probe, ath-miR399f and Thioflavin T (ThT) varies with the concentration of ath-miR399f (Horizontal axis is in logarithmic scale), the plot fitted with a linear regression.
Detailed Results
Extraction
Aim of this section:
We needed to determine an efficient system for the extraction of miRNA from raw plant tissue that is feasible both in timespan and in accessibility for the farmers. We had four methods to choose from, Whatman Paper, FTA Elute Paper, Salt Precipitation, and Isopropanol Precipitation. Each method had their strengths and weaknesses, and through a series of experiments, Isopropanol precipitation was determined to be the most viable method out of the four. Subsequently, a separate protocol, termed Farmer extraction, was developed that built off the principles of Isopropanol precipitation in a way that could better accomodate farmers. This included the use of a handheld centrifuge, which was tested to assess and optimise its performance.
Whatman Paper Results
Whatman Paper Results
FTA Elute Paper Results
FTA Elute Paper Results
Salt Precipitation Results
Salt Precipitation Results
Isopropanol Extraction Results
Isopropanol Extraction Results
Farmer extraction results
Farmer Extraction Results
Handheld centrifuge Results
Handheld centrifuge Results
To better assess and compare the extraction capabilities of the different extraction methods, RNA ScreenTape and RT-qPCR were also performed on the extracts obtained from each extraction method.
Comparisons With ScreenTape Results
Comparisons With ScreenTape Results
RT-qPCR Results
RT-qPCR Results
Detection
Aim of this section
The second part of our kit required us to develop a method of detecting miRNAs from our plant extracts. Three different detection methods were considered: two using RCA as a form of isothermal signal amplification (G quadruplex and BRET), and one without amplification based on Hybridisation. A box detector was also developed for farmers to detect fluorescent readouts using their smartphone.
RCA with BRET/G-Quadruplex Results
RCA with BRET/G-Quadruplex Results
Hybridisation results
Hybridisation results
Box Detector
Box Detector
Experiments were also conducted to optimise pre-hybridisation conditions.
qPCR Melt Curve Results
qPCR Melt Curve Results
UV Spectrophotometry Results
UV Spectrophotometry Results
Raw data plotted graphs from UV melting curves Plate 1
Raw data plotted graphs from UV melting curves Plate 1
- Red = 260 nm
- Blue = 280 nm
Raw data plotted graphs from UV melting curves Plate 2
Raw data plotted graphs from UV melting curves Plate 2
- Red = 260 nm
- Blue = 280 nm
Footnotes
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Renaud de la Faverie, A., Guédin, A., Bedrat, A., Yatsunyk, L. A., & Mergny, J. L. (2014). Thioflavin T as a fluorescence light-up probe for G4 formation. Nucleic acids research, 42(8), e65. https://doi.org/10.1093/nar/gku111 ↩
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Jiang, H.-X., Liang, Z.-Z., Ma, Y.-H., Kong, D.-M., & Hong, Z.-Y. (2016). G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection. Analytica Chimica Acta, 943, 114–122. https://doi.org/10.1016/j.aca.2016.09.019 ↩
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