Our goal
With the assistance of medical professionals, a blood sample is drawn and separated into serum, followed by RNA purification. Subsequently, the target circular RNA (circRNA) is amplified isothermally, and detection is performed using either a gold nanoparticle-based colorimetric assay or a lateral flow test, as illustrated in Figure 1. Both of the detection methods will be verified through experiments.
In the first method (Figure 1A), we employ rolling circle amplification (RCA) to amplify our target circRNA. RCA generates single-stranded complementary DNA (cDNA) with multiple copies of the circRNA sequence. Following this amplification step, testing is performed using 13 nm gold nanoparticles conjugated with probes.
In the second method (Figure 1B), we conduct reverse transcription-recombinase polymerase amplification (RT-RPA) to amplify our target circRNA. During RT-RPA, modified primers are included in the amplification process of circRNA, enabling the resulting cDNA products to be detected by lateral flow cassette, PCRD.
Figure 1: Colorectal Cancer Screening Workflow. After purifying circRNA, two detection methods are employed: A. Amplification through RCA, followed by gold nanoparticles colorimetric assay. B. Amplification through RT-RPA, followed by PCRD detection.
How to Prove our Concept?
We will divide our proof of concept into three parts as follows:
Separating Serum from Patients' Whole Blood and Purifying RNA:
Since our project is still under review by Chang Gung Medical Foundation Institutional Review Board for clinical trial application, currently we are unable to obtain blood samples from colorectal cancer patients to verify the process of circRNA isolation from blood. Thus, in order to confirm that our detection method of circRNA through liquid biopsy will function effectively after successfully accessing the purified circRNA from blood samples, this section will concentrate on a literature review to demonstrate the feasibility of circRNA isolation. [1]
Serum Preparation
Each participant is required to provide 8 mL of a blood specimen. After the collection of whole blood, allow the blood to clot by leaving it undisturbed at room temperature for 30 minutes, then remove the clot with a refrigerated centrifuge. The resulting supernatant is the designated serum. Following centrifugation, serum will be transferred into a clean polypropylene tube, ensuring that sample handling is maintained at 2-8°C. [2]
1. Exosome Isolation
Serum will be combined with ExoQuick exosome precipitation solution (System Bio-Sciences) according to the manufacturer's instructions. The resulting pellet is designated as an exosome. [1]
2. Exosomal RNA Extraction
Total exosomal RNA was extracted from the exosome pellets using the miRNeasy Serum/Serum Kit (Qiagen) according to the provided protocol. [1]
3. Ribonuclease R Treatment
Ribonuclease R (Thermo Fisher) will be added to the solution after exosomal RNA extraction. Ribonuclease R digests all linear RNAs but does not affect circular RNA structures, thereby increasing the proportion of circular RNA within the specimen.
Validation of Circularization
Due to legal constraints, we are unable to access patient samples for the purification of circRNA. Instead, we mimic circRNA in blood serum by circularizing RNA fragments synthesized from in vitro transcription (IVT).
Results of RNA circularization:
Our circularization process involves several steps, including sequence design, cloning, PCR, IVT, circularization and circRNA enrichment. Please refer to design page (Path A) for details.
The results of gel electrophoresis in the following image (Fig. 2) confirmed the success of our circularization process.
Figure 2. Agarose gel electrophoresis verifies the circularization process.
Lane 1: Low range RNA ladder
Lane 2: Linear RNA produced
by in vitro transcription. The presence of bands confirms the success of liner RNA synthesis,
and any remaining DNA templates are degraded by DNase I.
Lane 3: Circularization product. The sample contains circularization product and some linear RNAs remain
in the reaction.
Lane 4: Circularization
product + RNase R. Given the circRNA's resistance to RNA exonuclease activity, RNase R is
introduced to remove linear RNA. Therefore, the presence of bands indicated the success of
circularization.
Validation of Detection Method - Amplification Through RCA Followed by Gold Nanoparticles Based-Colorimetric Detection:
In this section, we will verify the followings:
(1) Verify whether RCA can amplify circRNA and generate long-repeated cDNA:
The gel electrophoresis results presented in the following image (Fig. 3) confirm the success of our RCA process.
Figure 3. Agarose gel electrophoresis verifies the RCA process.
Lane 1: 1000bp ssDNA ladder
Lane 2: CircRNA (202 ng) after 15 minutes RCA amplification
Lane 3: CircRNA (202 ng) after 30 minutes RCA amplification reaction
Lane 4: Low concentration of linear RNA (202 ng) after RCA 15 minutes amplification
Lane 5: Low concentration of linear RNA (202 ng) after RCA 30 minutes amplification
Lane 6: High concentration of linear RNA (2457.2 ng) after RCA 15 minutes amplification
Lane 7: High concentration of linear RNA (2457.2 ng) after RCA 30 minutes amplification
Compare lane 2 and 3 (using circRNA as template) with lane 4, 5, 6 and 7 (using linear RNA as template), the bands observed in lane 2 and 3 located at higher position than those in lane 4, 5, 6 and 7. This suggests the success of RCA, generating long-repeated cDNA.
(2) Verify gold nanoparticles based-colorimetric assay:
Unfortunately, we were unable to generate a sufficient amount of long-repeated cDNA for testing with probe-conjugated gold nanoparticles (AuNPs) detection before the competition. Therefore, in this section, we are initially adding RCA's no template control (NTC) solution to probe-conjugated gold nanoparticles and testing the relationship between the volume of MgCl2 added and the resulting color change.
Figure 4. The spectrum of adding MgCl2 to Probe conjugated-AuNP containing RCA solution.
Figure 5. The color of the Probe-Conjugated Gold Nanoparticles solution is influenced by the varying volumes of added MgCl2.
In Figure 4. we can observed a redshift in spectrum after adding MgCl2 to the Probe conjugated-AuNP solution with no target cDNA. Besides, in Figure 5, a significant color change can be observed when adding 6-8 µL of 0.21 M MgCl2.
Validation of Detection Method - Amplification through RT-RPA Followed by Lateral Flow Test:
(1) Amplify circRNA through RT-RPA
Figure 6. Gel electrophoresis of diluted RNA sample for RT-RPA. Lane 1: 100 bp DNA Ladder, Lane 2: RT-RPA using IVT product of circ_0101802 (385 ng/μL), Lane 3: A 10^4-fold dilution of the IVT product from Insert_0101802 (385 ng/μL); Lane 4: A 10^6-fold dilution of the IVT product from Insert_0101802 (385 ng/μL), Lane 5: A 10^8-fold dilution of the IVT product from Insert_0101802 (385 ng/μL), Lane 6: A 10^10-fold dilution of the IVT product from Insert_0101802 (385 ng/μL), Lane 7: A 10^12-fold dilution of the IVT product from Insert_0101802 (385 ng/μL), Lane 8: No Template Control.
Unfortunately, we were unable to test the amplification of circRNA by RT-RPA before the competition. However, we tested the limitation of linear RNA sample amplification through RT-RPA. We speculate that the limit of sample amplification for RT-RPA should be around 3.52*10^-10 ng/μL with a reaction time of 10 minutes. In the future, we aim to amplify the circRNA in patient blood.
In the future, if we have enough long-repeated cDNA, we will be able to test whether the probe-conjugated gold nanoparticles can recognize and bind to it, thereby maintaining a red color.
(2) Detection of RT-RPA-amplified products through PCRD.
The resulting RT-RPA products using linear RNA as template were conducted by lateral flow strip (PCRD). In the future, we aim to use RT-RPA to amplify circRNA and test it by PCRD.
| circ_0004771 | circ_0101802 | PCRD | |
|---|---|---|---|
| A. | + | + | 
|
| B. | － | ＋ | 
|
| C. | - | - | 
|
Diagram 1: The schematic diagram illustrates the different results obtained from the presence or absence of the target circRNA. (＋: added;－: not added)
In theory, the lateral flow strip should display only one control line when no target sequences added (as shown in diagram 1-C). However, our actual results indicate the presence of a test line during lateral flow test. To address this unexpected outcome, we have learned from a previous study [3] that primer dimers formed by the modified reverse and forward primers can lead to false-positive results. Therefore, we plan to design more specific primers in the future.
Based on the experimental results, we can conclude that when the target RNA is presented in the RT-RPA reaction, it can be successfully amplified and shows a positive result when further tested by PCRD. This observation is consistent with the expected outcome of our assay.
Reference
[1] Xie, Y., Li, J., Li, P., Li, N., Zhang, Y., Binang, H., Zhao, Y., Duan, W., Chen, Y., Wang, Y., Du, L., & Wang, C. (2020). RNA-Seq Profiling of Serum Exosomal Circular RNAs Reveals Circ-PNN as a Potential Biomarker for Human Colorectal Cancer. Frontiers in oncology, 10, 982. https://doi.org/10.3389/fonc.2020.00982
[3] Li, C.-J., Sun, H.-Q., Zhao, W.-X., Wang, X.-Y., Lin, R.-Z., & Yao, Y.-X. (2023). Rapid Assay Using Recombinase Polymerase Amplification and Lateral Flow Dipstick for Identifying Agrilus Mali, a Serious Wood-Boring Beetle of the Western Tianshan Mountains in China. https://doi.org/10.21203/rs.3.rs-2744727/v1