Sequence design → Cloning → PCR → In vitro transcription (IVT) → Circularization → CircRNA enrichment

    Cloning

Description

In this part, we ligase our insert with pJET1.2/blunt Cloning Vector and then transform it in DH5-alpha E. coli in order to produce abundant amounts of our target.

Data

Figure A1. Gel electrophoresis results of the colony PCR. lane 1: Bio-100 Mass DNA Ladder, lane 2: circ_0004771 colony 1, lane 3: circ_0004771 colony 2, lane 4: circ_0004771 colony 3, lane 5: circ_0004771 colony 4, lane 6: x, lane 7: x, lane 8: circ_0101802 colony 1, lane 9: circ_0101802 colony 2, lane 10: circ_0101802 colony 3, lane 11: circ_0101802 colony 4

Figure A1 shows the gel electrophoresis results of the colony PCR of the transformation product. We can find out that lane 4, 5, 9, and 10 have no unwanted bands, so we culture them and extract their plasmid for sequencing.

Figure A2. Sequence alignment of circ_0004771 colony 3 PCR product with circ_0004771 insert.

Figure A3. Sequence alignment of circ_0004771 colony 4 PCR product with circ_0004771 insert.

Figure A4. Sequence alignment of circ_0101802 colony 3 PCR product with circ_0101802 insert.

Figure A5. Sequence alignment of circ_0101802 colony 4 PCR product with circ_0101802 insert.

The sequencing results show that our product sequence is 99% similar to our insert, which indicates the success of the colony and it can be used for further experiments.

Achievement

Successfully ligase insert the vector and transform it into DH5-alpha E. coli.

    PCR

A. Insert PCR

Description

Insert PCR is the method we use for the rapid and large-scale production of Insert_0101802 and Insert_0004771. The resulting products can be used for circularization and experiment validation in other sections.

Data

Figure A6. PCR result of Insert_0004771. Lane 1 : 100bps ladder, Lane 2 : Insert_0004771

Figure A7. PCR result of Insert_0101802.Lane 1 : 50bps ladder, Lane 2,3 : Insert_0101802

Analysis

As shown in the Figure A6 and Figure A7, bright clear bands in the correct position can be seen, which indicates the successful amplifaction of Insert_0101802 and Insert_0004771 by Insert PCR.

B. Plasmid PCR

Description

Plasmid is produced in large quantities by DH5-alpha E. coli after cloning. In this part, plasmid is extract from the bacteria (for detailed please visit experiment-Plasmid extraction) and futher conduct plasmid PCR in order to rapidly produce large-scale production of Insert_0101802 and Insert_0004771. The resulting products can be used for circularization and experiment validation in other sections.

Data

Figure A8. Plasmid PCR result. Lane 1 : 100bps ladder, Lane 2, 4 : pjet1.2_0004771 , Lane 3, 5 : PCR of pjet1.2_0004771, Lane 6, 8 : pjet1.2_0101802 , Lane 7, 9 : PCR of pjet1.2_0101802

Analysis

As shown in the figure, we have successfully amplified pjet1.2_0101802 and pjet1.2_0004771 using Plasmid PCR. We will further purify by gel slices and us for further experiment.

    CircRNA synthesis (IVT & circularization & CircRNA enrichment)

In this process, we utilize the Insert 0101802 (Part) to undergo in vitro transcription (IVT), circularization, and circRNA enrichment. Due to time and resource constraints, circularizing two samples at once is not very efficient. Therefore, we use Insert 0101802 to initially validate the entire circRNA synthesis process's feasibility. Once we confirm the viability of the entire process, we can apply it to all samples to achieve our goal.

A. Verification of monophosphorylation and circularization product

Description

XRN-1 - verification of monophosphorylation

In order to verify the success of monophosphorylation, we utilize an enzyme, XRN-1. XRN-1 is an exoribonuclease enzyme that could degrade RNA sequences with a 5' monophosphate group. Therefore, RNA that is successfully monophosphorylated could be disintegrated by XRN-1. We run E-gel to compare monophosphorylation product with monophosphorylation product + XRN-1. If the monophosphorylation product + XRN-1 has a band as clear as the monophosphorylation product, then monophosphorylation is failed. Conversely, if the band is less distinct than that of the monophosphorylation product or no band is observed at all, it signifies successful monophosphorylation

RNase R - Eliminate linear form RNA

After circularization process, we are supposed to transform all the linear form RNA into circRNA. However, research related to circularization has not yet achieved 100% circularization rate. Therefore, in order to obtain pure circRNA, we use RNase R to eliminate failed circularization products, which are linear RNAs. After RNase R degrades the linear RNA, circRNA remains.

Figure A9. Verification of monophosphorylation and circularization product. Lane M: low range RNA ladder, Lane 1: Linear form RNA, Lane 2: Monophosphorylation product, Lane 3: Monophosphorylation product + XRN-1, Lane 4: Circularization product, Lane 5: Circularization product + RNase R, Lane 6: Circularization product + XRN-1, Lane7-10: Negative Control

Analysis

1. Lane1: Lane 1 is the sample of IVT product. After IVT, we obtained linear form RNA, as the above figure shows.
2. Lane 2 and Lane 3: Comparing lane 2 and lane 3, lane 2 obtained a brighter band than lane 3. After adding XRN-1, the monophosphorylated product has been degraded, leaving failed monophosphorylated samples. Therefore, the remaining band of lane 3 indicates the RNAs that have failed to be monophosphorylated. In summary, by contrast of these two lanes, we know that the efficiency of monophophorylation isn't 100%; there's still room for improvement.
3. Lane 4 and Lane 5: Comparing lane 4 with lane 5, lane 4 obtained a brighter band than lane 5. After adding RNase R to the circularized product, linear-form RNA could be degraded, leaving RNA with a circular structure. Therefore, lane 5 represents lane 4 sample eliminating linear form RNA, which means lane 5 are samples successfully circularized.
4. Lane 6: In order to reconfirm the success of circularization, we add XRN-1 to Circularization product. In circRNA, there's no monophosphorylated end to be degraded by XRN-1. Therefore, successfully circularized product should remain band after adding XRN-1.

Improvement

Sample bands in this E-gel experiment are slightly smear. We suspected that this smear phenomenon is caused by incomplete denature of samples. As a result, we revise denature condition from 65°C, 5 mins to 65°C, 15 mins.

Achievement

After conducting this experiment, we verify success of monophosphorylation and circularization, which proof that we're capable of synthesizing circRNA.

B. Comparing circularization efficiency of splint with different length

Description

In order to compare circularization efficiency of splint with different length, we carry out circRNA synthesis process using 14-mer splint, 16-mer splint, 18-mer splint, respectively. After running E-gel, splint with highest circularization efficiency obtained strongest band. Through this verification, we could utilize the splint with highest circularization efficiency in the following process.

Figure A10. Comparing circularization efficiency of splint with different length. Lane 1: low range RNA ladder, Lane 2: Control, Lane 3: Circularization product using 14-mer splint, Lane 4: Circularization product using 16-mer splint, Lane 5: Circularization product using 18-mer splint

Analysis

Lane 3, Lane 4, and Lane 5: Comparing lane 3, lane 4, and lane 5, we discovered that lane 4 has the strongest band. It indicates that using a 16-mer splint could maximize circularization yield.

Achievement

After conducting this experiment, we confirm utilizing 16-mer splint could maximize circularization yield.

C. Distinguish circRNA from linear form RNA

Description

Previous studies have mentioned that in gel electrophoresis, circRNA has higher running speed comparing to linear form RNA. This conclusion made use curious that whether our circularization product and linear form RNA could distinguish by their band position. Also, since we could merely distinguish band of ladder in E-gel results, we tried agarose gel instead. By utilizing agarose gel, we could clearly compare our sample bands to ladder band, for confirming size of samples.

Figure A11. Distinguish circRNA from linear form RNA. Lane 1: low range RNA ladder, Lane 2: Linear form RNA, Lane 3: Circularization product, Lane 4: Circularization product + RNase R, Lane 5: Circularization product + RNase R

Analysis

1. Lane 2 ~ Lane 5: According to the low-range RNA ladder, the size of the samples could be confirmed. IVT product, circularization product, and circularization product + RNase R are all around 300 bp, which conforms to our assumption.
2. Lane 2, Lane 4, Lane 5: Comparing lane 2 and lane 4, 5, we discovered that lane 2 band is higher than lane 4, 5. Previous research have mentioned that circular form RNA runs faster than linear form RNA. Since they obtained the same base pair, we suspect that it's the structure that affects their running speed. As a result, we conclude that Lane 4, Lane 5 is certainly circular RNA.

Achievement

After conducting this experiment, we confirm that all our samples obtained correct base pair as we expected. Furthermore, by comparing position of linear form RNA and circularization product, we verify the success of circularization.

D. Examine RNase R degradation ability

Description

In order to proof the degradation ability of RNase R is powerful enough to eliminate all the failed circularization product. We conduct following experiment, adding RNase R into linear form RNA product from in vitro transcription (IVT) process. We expected that RNase R could effectively degrade entire linear form RNA, which confirmed that it could also degrade entire failed circularization product after circRNA synthesis process.

Figure A12. Examine RNase R degradation ability. Lane M: low range RNA ladder, Lane 1: Linear form RNA, Lane 2: Linear form RNA + RNase R, Lane 3: Circularization product + RNase R, Lane 4-10: Negative Control

Analysis

1. Lane 1 and Lane 2: In order to make sure the degradation ability of RNase R is powerful enough to eliminate entire linear RNA in a circularized product, we add RNase R to IVT samples. After adding RNase R, we discovered that there's no band in lane 2, which indicates that RNase R has degraded the entire linear form of RNA. This result confirmed that RNase R could effectively eliminate redundant linear-form RNA in circularization samples, leaving pure circRNA.
2. Lane 3: After adding RNase R to the circularization product, there's a remaining band. This band is a sample that has been successfully circularized.

Achievement

After conducting this experiment, we confirm degradation ability of RNase R is powerful enough to eliminate all the failed circularization product

Sequence design → Gold nanoparticle (AuNPs) synthesis → Probe conjugated with Gold nanoparticle (AuNPs) → Probe conjugated with gold nanoparticle (AuNPs) detection

    Gold nanoparticle (AuNPs) synthesis

Description

To utilize a colorimetric assay for detecting colorectal cancer, our initial step involves synthesizing gold nanoparticles (AuNPs) of approximately 13 nm in diameter. Once we have successfully synthesized it, we can then proceed to conjugating probes onto these synthesized gold nanoparticles.

Data

Figure B1. Synthesis of gold nanoparticles in a two-neck flask

Figure B2. The spectrum of the gold nanoparticles exhibits a peak that is close to 520nm.

Figure B3. Examine gold nanoparticles under a scanning electron microscope (SEM) using the following conditions: voltage: 1500 kV, magnification: 230,000 folds.

Analysis

In previous studies, we discovered that 13 nm gold nanoparticles typically exhibit a peak at 520 nm in the spectrum[1]. To validate whether our self-synthesized gold nanoparticles are around 13 nm, we conducted a spectrum measurement. The results, shown in Figure B2, indicated a peak that closely aligns with 520 nm, providing strong evidence of successful synthesis. Additionally, we utilized a Scanning Electron Microscope (SEM) to examine gold nanoparticles. The results, shown in Figure B3, directly revealed that the gold nanoparticles have an approximate diameter of 13 nm, confirming that we have successfully synthesized gold nanoparticles of the desired size.

Achievement

1. Successfully synthesized 13 nm gold nanoparticles.

    Probe conjugated with gold nanoparticles (AuNPs)

A. Nanodrop Spectrum Analysis

Description

In this section, the probe will be conjugated with gold nanoparticles through an S-Au bond. Our probe is designed to detect the target cDNA generated by RCA. For more detailed design information, please refer to the Parts page.

Data

Figure B4 . A redshift shown in the spectrum of gold nanoparticles after conjugated with probes.

Analysis

Figure B4 revealed a redshift occurring in the spectrum of gold nanoparticles after conjugated with probes. According to previous studies [2], the redshift in the spectrum and a decrease in the optical density (O.D.) value are indicative of the successful conjugation of AuNPs with the probe.

Achievement

Successfully attached DNA probes to the surface of gold nanoparticles.

B. X-ray photoelectron spectroscopy (XPS) Analysis

Description

In addition to verifying the successful attachment of the probe to gold nanoparticles, we also employed X-ray Photoelectron Spectroscopy (XPS) to examine the presence of DNA probes on the surface of the gold nanoparticles. This involved examining elements associated with DNA probe and gold nanoparticles, such as Au, C, and N, and comparing the differences before and after probe modification on gold nanoparticles.

Figure B5. XPS results of gold nanoparticles and probe-conjugated gold nanoparticles.

Achievement

Successfully attached DNA probes to the surface of gold nanoparticles.

    Probe conjugated with Gold nanoparticle (AuNPs) detection

Description

Probe conjugated with gold nanoparticles will be used to conduct further detection of long reapeat cDNA sequence. For more detailed, please view Path C and Path D.

Sequence design → Cloning → PCR → Repeated insert sequence synthesis → Preparation of template for AELA-PCR → AELA-PCR → Probe conjugated AuNPs detection

    Repeated insert sequence synthesis

Description: We have added two restriction enzymes, BamH1 and HindIII, to cleave the PCR products. The resulting sticky ends at the 3' and 5' end of the inserts were then joined together using T4 DNA ligase, creating long-repeated insert sequences.

A. Insert digestion

Data

Figure C1. The agarose gel electrophoresis result of insert digestion. Lane 1: 100 bp DNA ladder. Lane 2: Insert_0004771 digestion product. Lane 3: Insert_0101802 digestion product.

Analysis

Lane 2: According to the DNA ladder, the base pair of digested
Insert_0004771
is between 200 bp and 300 bp, which conforms to our expectations.
Lane 3: According to the DNA ladder, the base pair of digested
Insert_0101802
is between 300 bp and 400 bp, which conforms to our expectations.

B. Insert ligation

Data

Figure C2. The agarose gel electrophoresis result of insert ligation. Lane 1: 1 kb DNA ladder, Lane 2: Insert_0004771 ligation product, Lane 3: Insert_0101802 ligation product.

Analysis

Lane 2: According to above figure, we discovered that the insert sequences are ligated in different fold numbers. Therefore, the result of agarose gel electrophoresis showed multiple bands with equal interval in Lane 2.
Lane 3: Similar to Lane 2, it also showed multiple bands with equal interval in Lane 3. The difference between the two lanes was that the band position of Insert_0101802 (Lane 3) was higher than Insert_0004771 (Lane 2).

Achievement

We successfully ligated each single piece of insert into a long-repeated dsDNA.

    Preparation of template for AELA-PCR

Description: We reintroduced the cloning process in an attempt to incorporate additional sequences and amplify the long-repeated insert sequences

A. Colony PCR

Data

Figure C3. The agarose gel electrophoresis result of colony PCR. Lane 1: 1kb DNA ladder, Lane 2 to Lane 5: the samples of colony PCR were picked from four different colonies.

Analysis

We observed the absence of bands in Lane 2, 4 and 5. In Lane 3, although there was a band present, its length fell between 500-1000 bp, shorter than our long-repeated insert sequence (approximately 1680 bp in total, 336 bp per piece). In summary, we did not achieve the expected amplification of the inserts.

B. Plasmid gel electrophoresis

Data

Figure C4. The agarose gel electrophoresis result of plasmid. Lane 1: 1 kb DNA ladder. Lane 2: pUC19 vector only. Lane 3: pUC19 vector ligated with long-repeated insert sequence.

Analysis

Lane 2: Only the pUC19 vector was loaded into Lane 2. The vector is around 2686 bp long.
Lane 3: We ligated the pUC19 vector (which is 2686 bp) with a long-repeated insert sequence (1680 bp in total, 336 bp per piece). The band marked with a red frame was around 4366 bp (vector 2686 bp + insert 1680 bp), which indicated the successful ligation.

Achievement

We successfully ligated each single piece of insert into a long-repeated dsDNA.

    AELA PCR

Since we failed to amplified the long-repeated insert sequences utilizing colony PCR as mentioned before. We could not further execute the experiments afterwards.

Sequence design → Cloning → PCR → IVT → Circularization → CircRNA enrichment → RCA → Probe conjugated with gold nanoparticles (AuNPs) detection

    Rolling Circle Amplification (RCA)

Description

Rolling Circle Amplification (RCA) is a kind of isothermal amplification method that can generate long-repeated complementary DNA (cDNA) of circRNA by reverse transcriptase. Compared to circular form RNA, linear RNA can only be reverse transcribed into complementary DNA (cDNA) with a length identical to that of the RNA template. Thus, in this experiment, we compared the amplification of linear form and circular form RNAs, to confirm whether RCA can generate long-repeated complementary DNA (cDNA) longer than the circRNA.

Data

Figure D1. Gel electrophoresis results verify the RCA process. Lane 1: 1000 bp ssDNA ladder, Lane 2: Circular RNA (202 ng) after 15 minutes RCA amplification, Lane 3: Circular RNA (202 ng) after 30 minutes RCA amplification, 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.

Analysis

In Figure D1, compared Lane 2 with Lane 4, we found that Lane 2 showed a higher position than Lane 4, indicating the successful generation of long-repeated complementary DNA (cDNA). By comparing Lane 4 to Lane 5, we concluded that conducting RCA for different time intervals does not significantly affect the results. Additionally, comparing Lane 7 to Lane 5, we observed that a higher sample concentration leads to a greater yield of reverse transcription products.

Achievement

Successfully generated a repeated cDNA sequence which is longer than the circRNA sequence for use in the gold nanoparticles colorimetric assay.

    Probe conjugated with gold nanoparticles (AuNPs) detection

Description

To determine whether a patient is infected with the disease, we can visually observe the color change of the gold nanoparticle solution under different conditions. According to previous studies, the addition of MgCl2 to the AuNPs solution induces aggregation and a color change (to purple). However, if the probes conjugated on the gold nanoparticles recognize and bind to the RCA product, the long-repeated cDNA sequence prevents AuNPs aggregation and also the color change (red) when MgCl2 is added.

Unfortunately, due to time constraints, we were unable to generate a sufficient amount of long-repeated cDNA for testing with Probe conjugated with gold nanoparticles (AuNPs) detection. Therefore, in this section, we are initially adding RCA NTC solution to Probe-Conjugated Gold Nanoparticles and testing the relationship between the volume of 0.21 M MgCl2 added and the resulting color change. In the future, if we have enough long-repeated cDNA, we will be able to test whether probe-conjugated gold nanoparticles can recognize and bind to it, thereby maintaining the color.

Data

Figure D2. The spectrum of adding MgCl2 to Probe conjugated-AuNP containing RCA solution.

Figure D3. The color of the Probe-Conjugated Gold Nanoparticles solution is influenced by the varying volumes of added 0.21 M MgCl2.

Analysis

In Figure D2. we can observed a red shift in spectrum after adding MgCl2 to the Probe conjugated-AuNP with no target cDNA. Besides, in Figure D3, a significant color change can be observed when adding 6-8 µL 0.21M MgCl2.

Achievement

We find out that 6–8 µL 0.21 M MgCl2 added to the Probe-Conjugated Gold Nanoparticles solution causes the color change and a redshift in spectrum.

Sequence design → Cloning → PCR → RPA → PCRD

    Recombinase Polymerase Amplification (RPA)

Description

Before RT-RPA, the ultimate amplification method of our choice, we adopted only RPA to confirm that the RPA primers we designed can work properly. After the reaction, gel electrophoresis was performed to check whether the band positions of the amplicons represented the same length as our inserts.

Data

Figure E1. RPA 2% agarose gel electrophoresis results. Lane 1: Bio-100 Mass DNA Ladder, Lane 2: Insert_0004771+primer 1 (RPA_0004771fw/rv_1), Lane 3 : NTC of Lane 2, Lane 4: Insert_0004771+primer 2 (RPA_0004771fw/rv_2), Lane 5: NTC of Lane 4, Lane 6: X , Lane 7: Insert_0101802+primer 1 (RPA_0101802fw/rv_1), Lane 8 : NTC of Lane 7, Lane 9: Insert_0101802+primer 2 (RPA_0101802fw/rv_2), Lane 10 : NTC of Lane 9.

Analysis

We confirmed the successful amplification of our target circRNA after RPA by verifying the length of the amplicons. However, the electrophoresis result showed that in RPA reaction of Insert_0004771, bands were observed in no template control (NTC) groups, futhermore, the control group and the sample-treated group had the same band positions. We suspect that the reagents might have been contaminated by the sample, leading to this result.

Achievement

The RPA primer we designed can function as expected, hence we could get into the next path—using linear RNA, which is the product of in vitro transcription (IVT), to conduct RT (reverse transcription)-RPA.

    PCRD

We conducted PCRD lateral flow test using amplicons produced from RT-RPA. (See Path F. PCRD)

Sequence design → Cloning → PCR → IVT → RT-RPA → PCRD

    RT-RPA

A. RT-RPA Analysis of Serially Diluted Linear RNA Samples

Description

Since we have ensured that the primers we designed can successfully work in RPA, we conducted a reverse transcription (RT)-RPA to amplify the RNA, which was obtained from in vitro transcription (IVT) of our insert (cDNA) (See Results—CircRNA Synthesis).

Data

Figure F1. Perform RT-RPA on a serial dilution of linear RNA samples. Lane 1: No Template Control (NTC); Lane 2: RT-RPA using IVT product of Insert_0004771 (68.2 ng/μL); Lane 3: A 10^2-fold dilution of the IVT product from Insert_0004771 (68.2 ng/μL); Lane 4: A 10^4-fold dilution of the IVT product from Insert_0004771 (68.2 ng/μL); Lane 5: A 10^6-fold dilution of the IVT product from Insert_0004771 (68.2 ng/μL); Lane 6: 100 bp DNA Ladder; Lane 7: No Template Control (NTC); Lane 8: RT-RPA of the IVT product from Insert_0101802 (97.8 ng/μL); Lane 9: A 10^2-fold dilution of the IVT product from Insert_0101802 (97.8 ng/μL); Lane 10: A 10^4-fold dilution of the IVT product from Insert_0101802 (97.8 ng/μL); Lane 11: A 10^6-fold dilution of the IVT product from Insert_0101802 (97.8 ng/μL).

Analysis

It can be confirmed that reverse transcriptase could effectively work together with RPA in a single step, as indicated by the clear and light band observed in our RT-RPA results.

Additionally, both of the no template control (NTC) using different pair of forward and reverse primers showed bright and clear band. The exist of band in (NTC) may caused by following reason:
(1) The contamination of DNA samples in RT-RPA reagents.
(2) The formation of self primer-dimer.
(3) The formation of hetero primer-dimer between forward and reverse primer.

B. PCR Analysis of RT-RPA Reagents for DNA samples Contamination

Description

Since we observed a band in the no template control (NTC) lane of RPA and RT-RPA experiment during gel electrophoresis, we suspected that contamination of the samples ( Insert_0004771 and Insert_0101802) may have contributed to this result. To confirm whether the reagents contained sample contamination, we conducted PCR tests on all RT-RPA reagents, including primers. The PCR tests were performed by utilizing PCR_0101802fw and PCR_0101802rv primers.

Data

Figure F2. Performance of PCR on the RT-RPA reagents to test for contamination. Lane 1: 100 bp DNA Ladder; Lane 2: Primer free rehydration buffer; Lane 3: RPA_0004771fw_1; Lane 4: RPA_0004771rv_1; Lane 5: RPA_0004771fw_2; Lane 6: RPA_0004771rv_2; Lane 7: RNase Inhibitor; Lane 8: dNTP; Lane 9: Magnesium Acetate (MgOAc); Lane 10: Nuclease-free water; Lane 11: ProtoScript®II Reverse Transcriptase.

Analysis

By conducting PCR tests on all RT-RPA reagents, the results revealed that bands occurred in Lane 2, Lane 4, and Lane 6, corresponding to the reaction using primer-free rehydration buffer, forward primer, or a reverse primer as a sample, respectively. These results indicate the primer-free rehydration buffer and the primers may have been contaminated.

C. RT-RPA Self-Primer Dimer Testing

Description

Since we observed a band in the no template control (NTC) lane of the RT-RPA experiment during gel electrophoresis, we suspected that the formation of self-dimer may have caused this result. Therefore, in this experiment, our objective is to add only one primer at once to assess the possibility of self-primer dimer formation during reaction of (NTC).

Data

Figure F3. Test of Self Primer Dimer. Lane 1: 100 bp DNA Ladder; Lane 2: RPA_0004771fw_1+RPA_0004771rv_1; Lane 3: RPA_0004771fw_2+RPA_0004771rv_2; Lane 4: RPA_0004771fw_1 only; Lane 5: RPA_0004771rv_1 only; Lane 6: RPA_0004771fw_2 only; Lane 7: RPA_0004771rv_2 only.

Analysis

Lane 4, Lane 5, Lane 6, and Lane 7 showed a faint band in the RT-RPA gel electrophoresis result, this may indicate the possibility of self-dimer formation. However, compared to Lane 1 and Lane 2, the positions of Lane 4, Lane 5, Lane 6, and Lane 7 are lower. Hence, it is less likely that the prominent band observed in the no template control (NTC) lane of the RT-RPA experiment is a primer's self-dimer.

D. RT-RPA Hetero-Primer Dimer Testing through NTC Sequencing

Description

To determine whether the band observed in the no template control (NTC) lane of the RT-RPA experiment is a result of sample contamination or the formation of hetero-dimer between forward and reverse primer, we conducted a repeat (NTC) of RT-RPA using forward primer RPA_0004771fw_1 and reverse primer RPA_0004771rv_1. Subsequently, we submitted the sample for Sanger sequencing at Genomics (a commercial genome sequencing company in Taiwan) and align the resulting sequence with our insert sequence by NCBI sequence alignment tool.

Data

Figure F4. Negative Template Control (NTC) of RT-RPA, using RPA_0004771fw_1 and RPA_0004771rv_1.

Figure F5. Alignment of the Sanger sequencing result with the sequence theory amplified by RPA_0004771fw_1 and RPA_0004771rv_1 primers, which corresponds to Insert_0004771.

Analysis

The result from Sanger sequencing (Figure F5.) indicated a 99% similarity to our target RNA sequence. We have concluded that the band observed in the no template control (NTC) lane is the result of contamination. Therefore, we ordered new primers and primer-free rehydration buffer for further experiments.

E. RT-RPA New Primer Testing

Description

Since the band in the no template control (NTC) lane of the RT-RPA experiment during gel electrophoresis is caused by contamination, we used new primers and primer-free rehydration buffer in this experiment.

Data

Figure F6. NTC of RT-RPA using different primers. Lane 1: 100 bp DNA Ladder; Lane 2: old RPA_0004771fw_2+RPA_0004771rv_2; Lane 3: new RPA_0004771fw_1+RPA_0004771rv_1; Lane 4: new RPA_0004771fw_2+RPA_0004771rv_2.

Analysis

Comparing lane 4 with lane 2, there was no band presented in the no template control (NTC) of RT-RPA using the new rehydration buffer and primers. This indicates that the contamination is coming from the rehydration buffer and primers.

F. Determining the Amplification Threshold of RT-RPA

Description

After successfully conducting the no template control (NTC) of RT-RPA, we can now proceed with further experiments. We have diluted our linear RNA sample (generated by IVT) and determined the limit of amplification using RT-RPA. Besides, in this experiment, we added 1.5 μL of both forward and reverse primers (10 μM each) and allowed them to react for 10 minutes.

Data

Figure F7. Gel electrophoresis of diluted RNA sample from 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 (NTC).

Analysis

As we seen in the gel electrophoresis results, the lower concentration of RNA sample, the lighter its band displays. Additionally, we can found out that the band is very light at diluting the sample solution to 10^-12 times (approximately 3.52*10^-10 ng/μL), thus we speculate that the limit of sample amplification for RT-RPA should be around 3.52*10^-10 ng/μL when we added 1.5 μL of both forward and reverse primer (10 μM) and react for 10 minutes.

G. Amplify circRNA by RT-RPA

Description

After successfully amplifying linear form RNA through RT-RPA, we switched our samples from linear form RNA to circular form RNA for RT-RPA.

Data

Figure F8. The gel electrophoresis results confirmed the success of the RT-RPA process for circRNA. Lane 1: 100 bp DNA Ladder, Lane 2: No Template Control, Lane 3: Linear form RNA, Lane 4: Circular form RNA

Analysis

As we seen in the gel electrophoresis results (Figure F8), both lane 3 and lane 4 have band at the same position, indicate we successfully amplify circRNA through RT-RPA.

    PCRD

Description

Our outcomes indicated that unnecessary oligonucleotides might form during RT-RPA reactions due to primer-dimers, which remained an unsolved problem. Despite that, we could still conduct lateral flow test with PCRD to confirm whether the customized lateral test strip we designed possessed the ability to catch the labeled-amplicons on it.

Lateral flow test was performed to detect RPA amplicons labeled with digoxigenin (DIG)/biotin and carboxyfluorescein (FAM)/biotin. PCRD cassette (Abington Health) (Figure F8) was used to visualize DIG/biotin or FAM/biotin-labeled amplicons.

RT-RPA sample (See Parts_Composite parts for more info)	5' modification of primer (See Sequence Design—RPA Primer to learn about primer design)	RT-RPA product	Corresponding line on the lateral flow strip	Antibody type
Insert_0004771	Forward primer: biotin Reverse primer: DIG	DIG/biotin labeled-amplicons	Test line 1	Anti-DIG capture antibody
Insert_0101802	Forward primer: biotin Reverse primer: FAM	FAM/biotin labeled-amplicons	Test line 2	Anti-FAM capture antibody
/	/	/	Control line (C)	Anti-mouse antibody

Fig F8. Composition of a PCRD lateral flow cassette. When the sample is applied to the cassette, the buffer flows down the strip, carrying the carbon-conjugated biotin antibodies and the amplicons with it. If the amplicons are present in the sample, they will bind to the anti-DIG monoclonal antibodies on the test line 1, or anti-FAM antibodies on the test line 2. The carbon particles will accumulate on the test line, making it visible. The rest of carbon-conjugated biotin antibodies will be captured on the C line, which can be served as control.

We utilized the product of RT-RPA directly to conduct lateral flow test. Three kinds of RNA samples were used in RT-RPA reaction: A. Insert_0004771 and Insert_0101802 mix, B. Insert_0101802 only, and C. No template control (NTC).

Data

Fig F9. Results of RT-RPA amplicons identification after 20 min incubation at 39 °C using a PCRD cassette: (A) RT-RPA amplicons of in-vitro transcription (IVT) products of Insert_0004771 and Insert_0101802 were added, two black test lines and a control line occured, revealing the successful binding of labeled-amplicons and antibodies on the strip, (B) The second test line corresponded with the RT-RPA product using in-vitro transcribed RNA of Insert_0101802 only, and (C) Only the control line presents when no template control (NTC) is added.

Analysis

In theory, there should be only a control line presented on the lateral flow strip when no target sequences are added (no template control), as shown in Figure F9. Nevertheless, our no template control (NTC) results showed a test line during the lateral flow test. To address this unexpected outcome, we learned from a previous study [3] and concluded that the formation of primer-dimers by the modified reverse and forward primers can lead to false-positive results. Therefore, we plan to design more specific primers in the future.

Our experimental results confirm that when target RNA is present in the RT-RPA reaction, it can be successfully amplified and show a positive result when further tested by PCRD. This observation is consistent with the expected outcome of our assay.

Achievement

We confirmed that the modified RPA primer can work through RT-RPA reaction, and made sure the reverse transcriptase works by conducting RT-RPA reaction. At last, confirming the modified primers we designed can generate labeled amplicons and successfully bind with corresponding antibodies. This was observed by examining the test lines on PCRD.

Unfortunately, due to time constraints, we were unable to complete this part before Wiki freeze. A significant portion of our time was devoted to determining the amplification threshold of RT-RPA, optimizing reaction times, primer concentrations, and addressing contamination issues. Consequently, amplify circRNAs utilizing RT-RPA will be a key focus part for our future work.

Reference

[1] Ali, M. M., Kanda, P., Aguirre, S. D., & Li, Y. (2011). Modulation of DNA-modified gold-nanoparticle stability in salt with concatemeric single-stranded dnas for colorimetric bioassay development. Chemistry - A European Journal, 17(7), 2052–2056. https://doi.org/10.1002/chem.201002677

[2] Li, F., Zhang, H., Dever, B., Li, X.-F., & Le, X. C. (2013). Thermal stability of DNA functionalized gold nanoparticles. Bioconjugate Chemistry, 24(11), 1790–1797. https://doi.org/10.1021/bc300687z

[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.