Part:BBa_K4664003
Padlock-miR-1274a
Padlock Padlock-miR-1274a is the corresponding padlock chosen for the miR-1274a biomarker.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Background
The padlock probe is essential to our experiments, especially the ligation and rolling circle amplification (RCA) steps. Both of these steps are included in the intermediary assays and our final MB-ERC2 (miRNA biomarker-based exponential RCP Cas12a/CRISPR) system. The general MB-ERC2 schematics is shown below in Figure 1.
Figure 1. MB-ERC2 Reaction Schematics.
Design
Padlock-1274a is a 57 bp-long linear single-stranded DNA (ssDNA). It is composed of three sections: the miR-1274a binding site (ligation module), crRNA recognition site (Cas12a detection module), and the intermediate sequence. Figure 2 is a visualisation of the padlock probe construct.
Figure 2. Padlock Probe Basic Construct
Results
The RT-qPCR results for miR-1274a (BBa_K4664001) and miR-223 (BBa_K4664000), respectively. We selected some of the RT-qPCR by-concentration graphs (Figures 3-5), a significantly lower fluorescence level for miR-1274a is already observable. A T-test to determine the statistical difference between NC-U6 and miR-1274a was also conducted. The P value was 0.7835, indicating no statistically significant difference. For more information see Table 1.
After analysis of our results, we excluded miR-1274a as our target of detection due to its poor performance with RT-qPCR, the ‘golden standard’ and conventional miRNA detection method. MiR-223 yielded significantly better results than miR-1274a and negative control U6, therefore we selected it as our primary target of detection.
Table 1. miR-1274a & U6 (100 pM).
Figure 3. By-concentration 100 pM.
Figure 4. By-concentration 1 pM.
Figure 5. By-concentration 20 fM.
References
Jin, J., Vaud, S., Zhelkovsky, A., Pósfai, J., & McReynolds, L. A. (2016). Sensitive and specific miRNA detection method using SplintR Ligase. Nucleic Acids Research, 44(13), e116. https://doi.org/10.1093/nar/gkw399
He, Y., Wen, Y., Tian, Z., Hart, N. T., Han, S., Hughes, S. J., & Zeng, Y. (2023b). A one-pot isothermal Cas12-based assay for the sensitive detection of microRNAs. Nature Biomedical Engineering. https://doi.org/10.1038/s41551-023-01033-1
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