Results Overview

In the journey of our iGEM project, our team has undertaken a series of experiments and simulations, pushing the boundaries of synthetic biology to address the detection of COPD.

While we reached notable milestones, our journey was filled with challenges. From the initial absence of a signal to pinpointing root causes, from system optimization to aligning our work with real-world applications, we confronted various hurdles. Each challenge offered invaluable learning experiences, strengthening our resilience and adaptability as researchers.


First, we conducted RT-qPCR trials, which will later be used as comparison groups for results obtained from MB-ERC2. Experiments were carried out for miR-1274a and miR-223 at concentrations of 100 pM, 10 pM, 1 pM, 500 fM, 100 fM, and 20 fM. Our goal was to determine the success of our selected miRNA biomarkers in an in vitro reaction.

RT-qPCR (Reverse Transcription Quantitative Real-time Polymerase Chain Reaction), is a combination of RT-PCR and qPCR methods, and is commonly employed for the detection and quantification of RNA. The procedure involves the enzyme reverse transcriptase converting total RNA or messenger RNA (mRNA) to complementary DNA (cDNA). This cDNA is then amplified and used in quantitative PCR (qPCR) or real-time PCR to detect specific targets. This PCR method utilises a number of fluorescent chemicals to quantify the amount of DNA at each cycle in real-time.

After the RT-qPCR trials, we excluded miR-1274a from future experiment plans due to its poor performance with RT-qPCR. U6 represents control in the following results figures.

U6 Trials: baseline/negative control for RT-qPCR

Figure 1 displays U6 RT-qPCR results at concentrations of 100 pM, 10 pM, 1 pM, 500 fM, 100 fM, and 20 fM.

Figure 1. RT-qPCR for U6.

miRNA RT-qPCR results

Figures 2 and 3 show the RT-qPCR results for miR-1274a and miR-223, respectively. A significantly lower fluorescence level for miR-1274a is already observable, and will be further demonstrated in the by-concentration charts.

Figure 2. RT-qPCR for miR-1274a.
Figure 3. RT-qPCR for miR-223.

Concentration-based Comparison of U6, miR-1274a, and miR-223

Figures 4 to 9, as shown below, are comparisons of U6, miR-1274a, and miR-223 fluorescence levels for the various concentrations used. miR-223 markedly outperforms both U6 and miR-1274a. It is evident that miR-1274a often yields performances similar to or even worse than the negative control U6 group. Thus, we excluded miR-1274a from future experiments.

Figure 4. 100 pM U6, miR-1274a, and miR-223.

A paired T-test was performed on miR-223 and U6. The two-tailed P value is under 0.0001, which is extremely statistically significant according to conventional criterion.

We also performed a paired T-test on miR-1274a and U6. P value equals 0.7835, meaning that by conventional criterion the difference of the two groups is not statistically significant.

For information about these two T-tests, see Tables 1 and 2.

Group miR-223 U6
Mean 2233.9849 2184.1433
SD 260.7545 177.8525
SEM 28.9727 19.7614
N 81 81
Table 1. miR-223 and U6 (100 pM)
Group miR-1274a U6
Mean 2182.8416 2184.1433
SD 199.8611 177.8525
SEM 22.2068 19.7614
N 81 81
Table 2. miR-1274a and U6 (100 pM)
Figure 5. 10 pM for U6, miR-1274a, and miR-223.
Figure 6. 1 pM for U6, miR-1274a, and miR-223.
Figure 7. 500 fM for U6, miR-1274a, and miR-223.
Figure 8. 100 fM for U6, miR-1274a, and miR-223.
Figure 9. 20 fM for U6, miR-1274a, and miR-223.

Intermediary Assays

Intermediary assays, or combinations of ligation, RCA, Cas12a, and qPCR, were carried out in order to investigate the effectiveness of RCA-Cas12a, on which our MB-ERC2 assay is based. Both the three-step and two-step assays are tandem combinations of RCA and Cas12a’s fluorogenic readout. However, in the three-step method (Figure 10a), ligation and rolling circle amplification (RCA) are separate, and the dye SYBR Green II is added (commonly used to stain RNA/ssDNA for qPCR analysis). In the two-step method (Figure 10b), RCA and ligation steps are combined, and the fluorogenic readout is solely from the fluorophore-quencher reporting system. Significant differences between the experimental groups and NC can already be observed.

Figure 10. Two step and three step assays mechanism.

Two Step Assay Results

Figures 11 and 12 are results from the two-step assay (RCA-ligation + CRISPR), with miRNA concentrations of 100 pM, 10 pM, 1 pM, 500 fM, 100 fM, and 20 fM. The two step assay is similar to our final MB-ERC2 system, except that the CRISPR step is still separate from the ligation-RCA step, and is non-isothermal. Markedly high results were obtained for most concentrations tested.

A paired T-test was performed for NC and 20 fM two step. The P value is less than 0.0001, indicating statistically insignificant difference.

Table 3 shows the data used in the T-test.

Group 20 fM NC
Mean 2121.4060 2014.9348
SD 34.3229 2.7662
SEM 6.8646 0.5532
N 25 25
Table 3. 20 fM vs. NC (Two Step).
Figure 11. Two-Step assay 10 pM & 100 pM.
Figure 12. Two-step assay 1 pM, 500 fM, 100 fM, and 20 fM result (x-axis is cycle, time per cycle is 10 minutes).

Three Step Assay

The three step assay is a tandem combination of ligation, RCA, and CRISPR. Experiments were performed at concentrations of 500 fM, 100 fM, and 20 fM, all of which yields significantly higher fluorescence levels than the NC.

A paired T-test was carried out for 20 fM vs. NC. P value is smaller than 0.0001, indicating statistically significant difference. Data is shown in Table 4.

Group 20 fM NC
Mean 2153.744 2041.2648
SD 51.6162 5.3643
SEM 10.3232 1.0729
N 25 25
Table 4. Three step 20 fM vs. NC.
Figure 13. Three-Step assay 500 fM, 100 fM, and 20 fM.


Our miRNA biomarker-based exponential RCA Cas12a/CRISPR (MB-ERC2) system combines rolling circle amplification with Cas12a cleavages. Figures 14-17 are results from MB-ERC2 experiments, a one-pot isothermal reaction system used to detect miRNA biomarkers at low, in vivo-level concentrations. Significant differences could be observed between the negative control and experiment groups, even for low concentrations such as 20 fM (close to miRNA concentrations in vivo). Results from various optimization experiments are also included (Figures 18-36). Please see more on the Experiments page.

Basic MB-ERC2 Reactions

Figures 14-16 are the basic reactions for MB-ERC2 (based on EXTRA-CRISPR from He et al. (2023)). miRNA concentrations include 1 nM, 100 pM, 10 pM, 1 pM, 500 fM, 100 fM, and 20 fM. Figure 17 is a comparison of no RNP vs. regular MB-ERC2 reactions, intended to demonstrate the importance of Cas12a cleavage in the reaction system.

A paired T-test was performed on EC-20 fM and NC. The P value is less than 0.0001, indicating an extremely statistically significant difference. Table 5 is data from the test.

Group EC 20 fM NC
Mean 2088.3783 2012.0552
SD 36.3071 4.5236
SEM 7.5705 0.9432
N 23 23
Table 5. T-test for EC 20 fM & NC.
Figure 14. EXTRA-CRISPR 100 pM, 1 nM (x-axis is cycle, time per cycle is 10 minutes).
Figure 15. EXTRA-CRISPR 500 fM, 1 pM, and 10 pM.
Figure 16. 100 fM & 20 fM.
Figure 17. NC RNP trials for 1 pM and 10 pM miRNA.

Optimization Reactions

Below are results from the various optimization experiments we performed in order to investigate the best concentrations of reagents involved in the MB-ERC2 system, and to optimise the sequence of the padlock.

We performed the following optimization experiments:

  1. Ribonucleoprotein (RNP) Concentration (Figures 21-23)
  2. Phi 29 Polymerase Concentration (Figures 18-20)
  3. Padlock Probe (PLP) Concentration (Figures 24-26)
  4. Reporter Concentration (Figures 27-29)
  5. Padlock Probe Sequence (Figure 35 & 36)
  6. BSA Concentration (Figure 33)
  7. Synthetic short-cuts (Figure 37)
  8. SplintR and T4 Ligase (Figures 31 & 32)
  9. SplintR ligase concentration (Figures 30 & 32)
  10. Buffer selection (Figure 34)

Phi 29 Polymerase Concentration

Phi29 polymerase is important in terms of facilitating the amplification in RCA (i.e. synthesis of the DNA strand complementary to the padlock). On the basis of our standard concentration (0.1 U/µl), we tested NC, 0.05 U/µl, 0.15 U/µl, 0.2 U/µl, and 0.5 U/µl

Figure 18. Phi29 Polymerase concentration optimization, 1 pM miRNA.
Figure 19. Phi29 Polymerase concentration optimization, 10 pM miRNA.
Figure 20. Final fluorescence levels for Phi29 concentration optimization.

Ribonucleoprotein (RNP) Concentration

Cas12a cleavages significantly influence the outcome of the reactions. Cis-cleavages are important for the initiation of secondary RCAs, thus promoting exponential amplification. Trans-cleavages are vital in terms of expression of the fluorescence signals. Thus, it is important to determine the optimal amount of RNP required to best enhance reaction kinetics. Our standard RNP concentration is 1 nM, thus, we tested the MB-ERC2 system with varied RNP concentrations of NC, 0.5 nM, 2.5 nM, 4 nM, and 5 nM.

Figure 21. Ribonucleoprotein concentration optimization, 10 pM miRNA.
Figure 22. RNP concentration optimization, 1 pM miRNA.
Figure 23. Ribonucleoprotein concentration optimization final values.

Padlock Probe (PLP) Concentration

The PLP-reporter ratio is vital to the experiment outcome (see Experiments page, Cas12a section). Padlocks are essential in the step of rolling circle amplification (RCA). Starting from the standard 100 nM, we set up trials with PLP concentrations of NC, 50 nM, 75 nM, 150 nM, and 200 nM.

Figure 24. Padlock Probe concentration optimization, 10 pM miRNA.
Figure 25. Padlock Probe (PLP) concentration optimization, 1 pM miRNA.
Figure 26. Padlock Probe concentration optimization final values.

Reporter Concentration

Our reporter is composed of a fluorophore linked to a quencher molecule. While the two are connected, the quencher absorbs energy from the fluorophore, thus preventing it from emitting fluorescence. But when cas12a trans-cleavage acts on the quencher-fluorophore and severs their link, the fluorophore would start giving out fluorescence. PLP-reporter ratio is essential to the results of the assay reaction, moreover, reporters generate the fluorescence signals on which our analytical hardware is solely based. We investigated concentrations of 0.5 µM, 1 µM, 2 µM, and 5 µM (standard is 10 µM).

Figure 27. Reporter concentration optimization, 1 pM miRNA.
Figure 28. Reporter concentration optimization, 10 pM miRNA.
Figure 29. Final fluorescences levels for reporter concentration optimization.

SplintR Ligase Concentration

SplintR ligase is extremely important for initiating the RCA process. We investigated concentrations of 10 U/20 µl, 20 U/20 µl, 25 U/20 µl, and 50 U/20 µl (standard 5 U/20 µl).

Figure 30. SplintR Ligase concentration optimization.

T4 and SplintR Ligases

According to Jin et al. (2016), SplintR ligase yields significantly better performance than T4 ligase. Thus, we designed this experiment, comparing 5 U/20 µl SplintR with 40 U/20 µl T4 ligase.

Figure 31. T4 vs. SplintR Ligase with 10 pM miRNA.
Figure 32. SplintR & T4 final fluorescence levels.

Bovine Serum Albumin Concentration

Different amounts of BSA were added to the reaction system. We used 0.1 mg/mL, 0.3 mg/mL, and 0.4 mg/mL (standard 0.2 mg/mL).

Figure 33. BSA concentration optimization.

Buffer Optimization

We investigated the following buffer combinations: 1) Buffer 2.1 (NEB) + BSA; 2) SplintR buffer + BSA, and; 3) Phi29 buffer + BSA. The SplintR/BSA combination yielded the best results.

Figure 34. Buffer selection.

Padlock Sequence Optimization

In order to determine the PLP sequence that yields the best reaction kinetics, we designed: 1) padlocks with the crRNA recognition site (responsible for binding with the crRNA in RNP and activating Cas12a cleavages) at different locations, and; 2) padlocks with single, double, and triple mismatches at the miRNA binding sites. We have 18 padlocks for miR-223 in total.

Figure 35. Padlocks 1-4 with 10 pM miRNA and optimal reaction system.
Figure 36. Padlocks M1-14 optimization.

Synthetic Short-cuts

We created a synthetic short-cut (short amplicon), which anneals to the padlock and initiates secondary RCA reactions. The short-cuts were then put in place of miRNA into the reaction system.

A paired T-test was also carried out for the 100 fM short-cut vs. NC. P value is less than 0.0001, meaning statistically significant difference. Data is shown in Table 6.

Group Short-cut 100 fM NC
Mean 2332.2464 2050.9732
SD 102.5636 2.9386
SEM 20.5127 0.5877
N 25 25
Table 6. T-test for 100 fM short-cut vs. NC.
Figure 37. Synthetic short-cuts.


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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.

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