Sequence Design
All necessary DNA sequences for each step must be designed before beginning the experiment. Please refer to the Parts for more details.
Cloning
Ligation
Equipment
1. Dry bath
Consumables
1. 2X Reaction Buffer (Thermo Fisher Scientific)
2. pJET1.2/blunt Cloning Vector (50 ng/µL) (Thermo Fisher Scientific)
3. T4 DNA Ligase (400,000 U/mL) (New England Biolabs)
4. Sample (insert)
5. Nuclease-free water (Protech Technology)
Protocol
1. Set up the ligation reaction on ice. Vortex briefly and centrifuge for 3-5 s.
2. Incubate the ligation mixture at room temperature (22 °C) for 30 min.
3. Use the ligation mixture directly for transformation.
Transformation
Equipment
1. Dry bath
2. Disposable cell spreaders
3. Incubator
Consumables
1. DH5-alpha E. coli (New England Biolabs)
2. Sample (ligation product)
3. LB medium plate with ampicillin
4. LB medium with ampicillin (0.1 mg ampicillin / 1 mL LB)
Protocol
1. Pipette 100 µL of DH5-alpha E. coli from the stock and then aliquot 50 µL into another
eppendorf tube. Place the tube on ice.
2. Add 5 µL of ligation product to the aliquoted DH5-alpha E. coli, flick the tube, and
incubate it on ice for 25 minutes, flicking gently every ten minutes.
3. Place both tubes in a 42°C dry bath for 45 seconds.
4. In a laminar flow hood, add 1 ml of LB medium with ampicillin to each tube, and incubate
them
at 37°C with shaking for 1 hour.
5. Take 100 µL of the mixture and spread it evenly on a LB medium plate with ampicillin
using
a disposable cell spreaders.
6. Incubate the LB medium plate with ampicillin in an incubator at 37°C for 16 hours.
Colony PCR
Equipment
1. Thermocycler
2. Culture tube
3. Dry bath
4. Incubator
Consumables
1. LB medium with ampicillin (0.1 mg ampicillin / 1 mL LB)
2. 2X Master Mix (Protech Technology)
3. pJET1.2 Forward Sequencing Primer, 10 µM (Thermo Fischer Scientific)
4. pJET1.2 Reverse Sequencing Primer, 10 µM (Thermo Fischer Scientific)
5. Sample (transformation product)
6. Nuclease-free water (Protech Technology)
Protocol
1. Use pipette tips to pick colony, each petri dish choose 4 colonies.
2. Put the colony in 200 µL of LB with ampicillin (0.1 mg ampicillin / 1 mL LB).
3. Shake in a 37°C incubator for 2 hours in preparation for colony PCR.
4. Add the following reagents to PCR tube.
5. React in a thermocycler using following setting.(x 35 cycles)
6. Perform 1.5% agarose gel electrophoresis.
7. Identify samples with the correct sequence length and no unwanted bands. Then transfer 10
µL
succefully transformed bacteria liquid to 5 mL LB with ampicillin in culture tube.
8. Shake in a 37°C incubator for 16 hours.
Plasmid extraction
Equipment
1. Dry bath
2. Centrifuge
Consumables
1. 50% Glycerol
2. Mini Plus Plasmid DNA Extraction System (Viogene)
3. Sample (colony PCR product)
Protocol
1. Take 500 µL of a 16-hour bacterial culture and use it for bacterial cryopreservation. Mix the
500 µL bacterial culture with 500 µL of glycerol in an Eppendorf tube, then store it at -80
°C.
2. Take 1 mL of the remaining 4.5 mL bacterial culture at a time and centrifuge it 1 min at
10,000rpm in Eppendorf tubes. After each centrifugation, discard the supernatant using a
pipette.
3. Add 200 µL Solution1 and pipette it until the cells are completely resuspended.
4. Add 200 µL Solution2 and mix by gently inverting the capped tube 5-6 times. Incubate at
room temperature for 5 min.
5. Add 300 µL Solution3 and mix by gently inverting the capped tube 5-6 times. Incubate at room
temperature for 3 min.
6. Centrifuge it at 10,000 rpm for 5 minutes. You will observe the formation of a dense, white
pellet either along the side or at the base of the tube.
7. Load the spin column into a collection tube, transfer the supernatant from step 6 directly to
spin column, spin for 1 min at 10,000 rpm.
8. Take out the spin column from the collection tube, dispose of the liquid that flows through
it, reinsert the spin column, and then apply 700 µL of washing solution. Centrifuge for 1
minute.
9. Take out the spin column from the collection tube, pour away the liquid, reinsert the spin
column, and then apply 700 µL of washing solution. Centrifuge for 1 minute.
10. Dispose of the filtrate and then centrifuge at 10,000 rpm for 3 minutes to eliminate any
remaining traces of ethanol. Incubate the spin column in a 60 °C oven for 8 minutes.
11. Remove the spin column and place the column in a new Eppendorf tube.
12. Add 50 µL free water into the column. Elute DNA by centrifugation for 1 min and store the
eluted DNA at -20 °C.
All-aspect PCR
Plasmid PCR
Equipment
1. PCR tubes
2. Thermocycler
Consumables
1. 2X Master Mix (Protech Technology)
2. Nuclease-free water (Protech Technology)
3. Forward primer (10 μM)
4. Reverse primer (10 μM)
5. Sample (plasmid)
6. Gel/PCR DNA Isolation System (Viogene)
Protocol
1. Add the following reagents to PCR tube.
| Reagents | Amount |
| 2X Master Mix | 10 μL |
| Foward primer | 0.5 μL |
| Reverse primer | 0.5 μL |
| Sample | 2 μL |
| Nuclease-free water | to 20 μL |
| Total | 20 μL |
2. React in a thermocycler at following setting.
| Stage | Temperature | Time | |
| Denaturation | 95 °C | 5 min | |
| Denaturation | 95 °C | 30 sec | 35 cycles |
| Annealing | 61 °C | 30 sec | |
| Extension | 72 °C | 1 min | |
| Extension | 72 °C | 7 min | |
| Cooling | 4 °C | ∞ |
3. Run the plasmid PCR DNA on a 1.5 % agarose gel.
4. Conduct gel purification using Gel/PCR DNA Isolation System followed the provided
protocol.
5. Measure a DNA concentration of the purified product by NanoDrop.
Insert PCR
Equipment
1. PCR tubes
2. Thermocycler
Consumables
1. Pfu Buffer (5 U/µL) (Protech Technology)
2. Pfu Polymerase (5 U/µL) (Protech Technology)
3. Forward primer (10 μM)
4. Reverse primer (10 μM)
5. dNTP (10 mM) (Thermo Fischer Scientific)
6. Sample (template)
7. Gel/PCR DNA Isolation System (VIOGENE)
Protocol
1. Add the following reagents to PCR tube.
| Reagents | Amount |
| Pfu Buffer | 5 μL |
| Foward primer | 1 μL |
| Reverse primer | 1 μL |
| dNTP | 1 μL |
| Pfu pol | 0.25 μL |
| Template (plasmid) | 1 μL |
| Nuclease-free water | to 50 μL |
| Total | 50 μL |
2. React in a thermocycler at following setting.
| Stage | Temperature | Time | |
| Denaturation | 95 °C | 5 min | |
| Denaturation | 95 °C | 1 min | 35 cycles |
| Annealing | 61 °C | 30 sec | |
| Extension | 68 °C | 1 min | |
| Extension | 68 °C | 10 min | |
| Cooling | 4 °C | ∞ |
3. Run the insert PCR DNA on a 1.5 % agarose gel.
4. Conduct gel purification using Gel/PCR DNA Isolation System followed the provided
protocol.
5. Measure a DNA concentration of the purified product by NanoDrop.
In Vitro Transcription (IVT)
Equipment
1. Dry bath
2. NanoDrop Spectrophotometers
Consumables
1. Nuclease-free water (Protech Technology)
2. 10X RNA Pol Reaction Buffer (New England Biolabs)
3. NTP (100 mM) (Thermo Fisher Scientific)
4. RNase Inhibitor (40,000 U/mL) (New England Biolabs)
5. DTT (0.1 M) (Thermo Fisher Scientific)
6. T7 RNA Polymerase (50,000 U/µL) (New England Biolabs)
7. GeneJET RNA Cleanup and Concentration Micro Kit (Thermo Fischer Scientific)
Protocol
1. Mix the following reagents at room temperature. The T7 RNA polymerase should be added last.
| Reagents | Amount |
| 10X RNA Pol Reaction Buffer | 2 μL |
| NTP | 0.1 μL for each |
| RNase Inhibitor | 1 μL |
| DTT | 1 μL |
| cDNA (1 μg) | Variable |
| T7 RNA poly | 2 μL |
| Nuclease-free water | to 20 μL |
| Total | 20 μL |
2. Incubate at 37 °C for 1 hr.
3. In order to eliminate DNA template used for in vitro transcription (IVT) process, we added DNase
I for DNA degradation.
DNase I calculation method:
a. According to the product information page of Thermo
Fisher[1] two units of DNase I are required to degrade 1 μg of DNA. As a
reference, we could
calculate the amount of DNA to be digested in order to determine the required amount of DNase I
to be added.
b. In the process of in vitro transcription (IVT), we utilize
a fixed amount of DNA template. The weight of the DNA we used is 500 ng. 500 ng = 0.5 μg of DNA;
therefore, the required DNase I is 1 unit. Since the concentration of DNase I is 1 U/μL, the
amount of DNase I needed in this process is 1 μL.
4. After adding DNase I, incubate the mixture at 37 °C for 15 minutes.
5. Follow the DNase I clean-up protocol of GeneJET RNA Cleanup and Concentration Micro Kit
(Thermo Fischer Scientific)
6. Use NanoDrop to measure the concentration of sample.
Circularization
Equipment
1. PCR tubes
2. Thermocycler
3. NanoDrop Spectrophotometers
Consumables
1. Sample (IVT product)
2. 18 bp Splint (25 µM)
3. 16 bp Splint (25 µM)
4. 14 bp Splint (25 µM)
5. Pyrophosphatase (1 U/µL)(Thermo Fischer Scientific)
6. T4 RNA ligase 2 (New England Biolabs)
7. T4 RNA ligase 2 Reaction Buffer (New England Biolabs)
8. DNase I (1 U/µL) (Thermo Fischer Scientific)
9. GeneJET Plasmid Miniprep Kit (Thermo Fischer Scientific)
10. RNase-free water (Protech Technology)
Protocol
Monophosphorylation
1. In order to monophosphorylate the RNA sample from above-mentioned process, pyrophosphatase is
the enzyme required. According to product page of Thermo Fisher[1], one unit of
pyrophosphatase
enzyme could catalyzes 1 μmol of inorganic pyrophosphate. As a reference, we could calculate the
mole of RNA to be monophosphorylated, in order to determine the required amount of
pyrophosphatase to be added.
2. The total amount of product could be calculated by following formulas:
a. Mass of RNA sample: Concentration of RNA × Total volume required to be monophosphorylated = Mass
of RNA sample
b. Mole of RNA sample: (Mass of RNA sample) / (Molecular weight of sequence) = Mole of RNA
sample
c. Pyrophosphatase to be added: Mole of RNA sample (the unit should be convert to μmol) = Unit of
pyrophosphatase enzyme to be added
3. Incubate the RNA sample that has added monophosphatase at 25°C, for 30 min.
4. Verification of Monophosphorylation:
a. 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.
b. Add the following components for verification:
The sample required is 1 μg, so the volume of sample to be added could be calculate as 1
μg/concentration of monophosphorylated sample.
c. Incubate for 60 minutes at 37 °C.
| Reagents | Amount |
| XRN-1 | 1 μL |
| 10X reaction buffer | 1 μL |
| Sample | 1 μg |
| RNase-free water | to 10 μL |
| Total | 10 μL |
5. Follow the protocal of GeneJET Plasmid Miniprep Kit (Thermo Fischer Scientific).
6. Use NanoDrop to measure the concentration of sample.
Formation of dsRNA
1. We utilize T4 RNA ligase 2 to back-splice the linear RNA, which requires forming dsRNA using
ssRNA combined with splint.
2. Splint should be added, and the volume could be calculated by following formulas:
a. Calculate the mole of our ssRNA product: Concentration of RNA × Volume of ssRNA product ÷
Molecular weight of the sequence = Mole of ssRNA product (the unit should be convert to pmol)
b. Calculate the mole of splint should be added: Since the molar ratio of splint : ssRNA
should be 2 : 1, the mole of splint = mole of ssRNA × 2.
c. Calculate the volume of splint should be added: The mole of splint ÷ Concentration of the
splint = Volume of splint
3. Incubate the mixture at 95°C for 2 min and turn off thermocycler to let it cool down for 1.5 hr.
Back splicing
1. Calculate the total volume of dsRNA product from last step: Volume of splint + Volume of ssRNA =
Volume of dsRNA product.
2. Calculate the concentration of dsRNA mixture: Mole of ssRNA product ÷ Volume of dsRNA product =
Concentration of dsRNA mixture
3. Calculate the volume of dsRNA product required for circularization: The ideal mole of
circularization reagent is 20 µmol. So we could calculate the required volume of dsRNA product by 20
µmol ÷ Concentration of dsRNA mixture.
4. Add the components as following chart:
| Reagents | Amount |
| 10X T4 RNA ligase 2 Reaction Buffer | 2 μL |
| T4 RNA ligase 2 | 1 μL |
| dsRNA product | Variable |
| RNase-free water | to 20 μL |
| Total | 20 μL |
5. Incubate the mixture at 25°C for 1hr.
6. In order to eliminate splint DNA used in circularization process, we add DNase I for DNA
degradation.
a. Two units of DNase I is required to degrade 1 μg of DNA. As a reference, we could calculate
the amount of splint DNA to be digested, in order to determine the required amount of DNase I to
be added.
b. Calculate the mass of the added splint DNA : In original tube of splint, it indicates the
volume and mass of splint. By these two value, we could calculate the mass of splint (µg) per
volume (µL). Then the volume of added splint DNA could be calculate by mass (µg) of splint per
volume (µL) × Volume of splint added = Mass of added splint DNA (µg)
c. Calculate the DNase I should be added: Mass of added splint DNA (μg) × 2 = Unit of DNase
should be added.
7. Follow the DNase I clean-up protocol of GeneJET RNA Cleanup and Concentration Micro Kit (Thermo Fischer Scientific).
CircRNA enrichment
Equipment
1. Dry bath
2. E-Gel™ Power Snap Electrophoresis System (Invitrogen™)
Consumables
1. RNase R (10 U/μl) (abcam)
2. RNase inhibitor (40,000 U/ml) (New England Biolabs)
3. 10X RNase R Reaction Buffer (abcam)
4. Nuclease-free water (Protech Technology)
5. Low range RNA ladder or Century™-Plus RNA Markers (Thermo Fischer Scientific)
6. 2X RNA loading dye (New England Biolabs)
Protocol
RNase R degradation
1. RNase R - Eliminate linear form RNA:
After the circularization process, we are supposed to transform all the linear RNA into circRNA. However, research related to circularization has not yet achieved a 100% circularization rate. Therefore, in order to obtain pure circRNA, we use RNase R to eliminate failed circularization products, which are linear RNAs, to get a higher percentage of circRNA for further experiments.
2. Calculate RNase R required for degrading linear RNA:
a. According to product information page of Abcam[3], one unit of RNase R could
convert 1 µg of
poly(A) into nucleotides. As a reference, we should calculate the total mass of RNA for further
degradation.
b. Mass of total RNA sample: Concentration of RNA (calculated by previous step by Nanodrop)
× Total volume of RNA = Mass of total RNA sample.
c. Unit of RNase R to be added: Mass of total RNA sample (the unit should be convert to μg) =
Unit of RNase R to be added
d. Volume of RNase R to be added: According to Abcam website[4],
concentration of RNase R is 10 U/µL, so the volume of RNase R to be added = Unit of RNase R÷10.
3. Add the following components:
| Reagents | Amount |
| RNA sample | 1 μL to 10 μL |
| RNase R | 0.2 μL to 2 μL (according to RNA sample) |
| RNase Inhibitor | 0.5 μL |
| 10X RNase R Reaction Buffer/td> | 2 μL |
| Nuclease-free water | to 20 μL |
| Total | 20 μL |
4. Incubate the mixture at 37°C for 2hr.
5. Follow protocol of GeneJET Plasmid Miniprep Kit (Thermo Fischer Scientific).
E-gel EX electrophoresis
Sample preparation
1. Prepare the sample and mix it with dye in the ratio of one to one.
2. There are ten wells in one E-Gel EX cassette. If the number of loading samples is less than ten,
the empty wells should be filled with water and dye in a one-to-one ratio.
3. The RNA ladder with dye and samples with dye should all be denatured before loading them into
E-Gel EX cassette wells. For RNA ladder denaturation, it has to be denatured at 90 °C for 2 minutes.
While for the denature of the sample, it has to go under 65 °C for 15 minutes.
Operation
1. Open up the Thermo Fisher Scientific E-Gel EX package.
2. Take out the E-Gel EX cassette and place it into the E-Gel Power Snap System. Make sure the
electrode is placed correctly.
3. Make sure that the lid of the E-Gel EX cassette has been removed before loading the sample.
4. After the sample has all been loaded, close the lid of the E-Gel Power Snap System.
5. Turn on the E-Gel Power Snap System and set the options and parameters according to your
experiment.
6. After the setting is done, press start to run gel.
7. The results can be observed under the E-Gel Power Snap System hood simultaneously during the run.
AuNPs synthesis
Equipment
1. Two-neck flask
2. Liebig condenser
3. Heating mantle
4. Hot plate/magnetic stirrer
5. Thermometer
6. Automatic pipette
7. Stir Bar
8. 50 mL centrifuge tubes
Consumables
1. HAuCl4 powder
2. Deionized water
3. 1% Sodium citrate
4. Aluminum foil
Protocol
1. 3.1 mM HAuCl4 5.69 mL + 14.11 mL deionized water mixed in two-neck flask, put a
magnetic
stir bar in the two-neck flask.
2. Heat it in a heating mantle until boiling (130 °C, ocean sand, approximately 10 min), while
gently stirring it (60 rpm). *Note: Put the condenser at one of the neck, the other neck closed
by a stopper (Aluminum
foil).
3. Turn the stir to 400 rpm.
4. Add 2.4 mL 1% sodium citrate in the two-neck flask (remember to add it all at once!).
5. When the color changes into burgundy red, keep heating for 10-15 min.
6. Remove the heating mantle, continue stirring.
7. After cooling down, pour all into a centrifugation tube.
8. Test it with NanoDrop spectrophotometer, its peak wavelength will be close to 520 nm.
SEM/EDS Analysis
Equipment
1. Diamond glass cutter
2. 250 mL beaker
3. Ultrasonic cleaner
4. Laboratory oven
5. Vacuum dryer
6. SEM-IT800
7. IT800-EDS
Consumables
1. Silicon wafer
2. Dish detergent
3. Ethanol
4. Acetone
5. Petri dish
Protocol
Sample Preparation
1. Cut the silicon wafer to approximately 0.4 cm*0.4 cm square with a diamond glass cutter.
2. Put the silicon wafer into a clean beaker. Wash the silicon wafer with diluted dish detergent
in an ultrasonic cleaner for 12 minutes.
3. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with ddH2O in an
ultrasonic cleaner for 12 minutes. Repeat this step three times.
4. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with ethanol in an
ultrasonic cleaner for 12 minutes.
5. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with acetone in an
ultrasonic cleaner for 12 minutes.
6. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with ddH2O in an
ultrasonic cleaner for 12 minutes.
7. Transfer it to a clean petri dish. Dry it in an oven.
8. Drop 1 µL of nanoparticles sample on a silicon wafer.
9. Put it in a vacuum dryer for 1-2 hours, confirm the sample is completely dry.
Analysis
1. Using SEM-IT800 to observe the shape of gold nanoparticles (AuNPs) and measure their diameter. Additionally, utilizing IT800-EDS to analyze the elemental composition and distribution in the sample.
Probe conjugated AuNPs
[Option 1] Probe conjugated-AuNP synthesis
Equipment
1. Disposable scintillation vials
2. Centrifuge
3. 1.5 mL microcentrifuge tube
Consumables
1. Gold nanoparticles (3 mL, 36 pmol, O.D. = 3.24)
2. Deionized water
3. 1M Tris-HCl (pH 7.5)
4. 1 M NaCl
5. 10 mM TCEP
6. DNA probes (1 mM)
7. ddH2O
8. 500 mM acetate buffer (pH 5.2)
9. 1M NaCl
10. Binding buffer (3 mL, 20 mM Tris-HCl, pH 7.5, 100 mM NaCl)
Protocol
1. Soak two disposable scintillation vials (20 mL volume) in 12 M NaOH for 1 hr at room
temperature. Rinse the vials with copious amounts of deionized water.
2. Prepare 10 mM TCEP
3. Pipette 1.8 µL of 1 mM DNA, 7.2 μL ddH2O into a 1.5 mL microcentrifuge tube.
4. Add 1 μL of 500 mM acetate buffer (pH 5.2) and 1.5 µL of 10 mM TCEP to each tube to activate
the thiol-modified DNA. Incubate the sample at room temperature for 1 hr.
5. Gold nanoparticles (3 mL, O.D.= 3.24) was incubated at room temperature with the thiol-modified
DNA oligonucleotide (1800 pmol) for 24 hr.
6. Tris-HCl (30 μL, 1 M, pH 7.5; all pH values were measured at 23 °C) and an aqueous solution
of NaCl (270 μL, 1 M) were added, mixed, and incubated at RT for another 24 h.
7. Tris-HCl (15 μL, 1M, pH 7.5) and aqueous solution of NaCl (50 μL, 1M) were added and further
incubated for 24 h.
8. The aged AuNPs were isolated by centrifugation at 15,000 g at RT for 10~30 mins, re-suspended
in the binding buffer, and stored at RT.
[Option 2] Probe conjugated-AuNP synthesis
Equipment
1. Disposable scintillation vials
2. Centrifuge
3. 1.5 mL microcentrifuge tube
Consumables
1. 1 mM DNA
2. TCEP (10mM)
3. 12 M NaOH
4. 500 mM acetate buffer (pH 5.2)
5. Gold nanoparticles (O.D.=3.5)
6. Nuclease-free water (Protech Technology)
7. 500 mM Tris acetate (pH 8.2) buffer
8. 1 M NaCl
9. Binding buffer (20 mm Tris-HCl, pH 7.5, 100 mm NaCl)
Protocol
1. Soak two disposable scintillation vials (20 mL volume) in 12 M NaOH for 1 hr at room
temperature. Rinse the vials with a copious amount of deionized water.
2. Prepare 10 mM TCEP
3. Pipette 9 µL of 1 mM DNA probe into a 1.5 mL microcentrifuge tube.
4. Add 1 µL of 500 mM acetate buffer (pH 5.2) and 1.5 µL of 10 mM TCEP to each tube to activate
the thiol-modified DNA. Incubate the sample at room temperature for 1 hr.
5. Transfer 3 mL of the already prepared gold nanoparticles (O.D. value 3.5) to each of the two
NaOH-treated glass vials, and then add the TCEP-treated thiol DNA with gentle shaking by hand
(or pipetting), and then mix the solution by stir bar for 16 hr (avoid light).
6. Add 30 µL of 500 mM Tris acetate (pH 8.2) buffer dropwise to each vial with gentle hand
shaking. The final Tris acetate concentration is 5 mM.
7. Add 300 µL of 1 M NaCl dropwise to each vial with gentle hand shaking. Store the two vials in
a drawer for at least another day before use (avoid light).
8. Centrifuge at 10,000 rpm, 20 mins. (The centrifugation condition might change.)
*Note: Adjust the centrifugation condition (duration and/or speed): The supernatant after
centrifugation should be as transparent as possible, instead of a pink color. Also, there
shouldn't be any black dots remaining on the eppendorf's wall, which indicates there's
unfavorable aggregation of gold nanoparticles.
9. Remove the supernatant, resuspend with 1 mL of binding buffer.
X-ray photoelectron spectroscopy (XPS) Analysis
Equipment
1. Diamond glass cutter
2. 250 mL beaker
3. Ultrasonic cleaner
4. Oven
5. Vacuum dryer
6. High resolution X-ray Photoelectron Spectrometer (ULVAC-PHI, PHI Quantera Ⅱ)
Consumables
1. Silicon wafer
2. Dish detergent
3. Ethanol
4. Acetone
5. Petri dish
Protocol
Sample Preparation
1. Cut the silicon wafer to approximately 0.8 cm*0.8 cm square with a diamond glass cutter.
2. Put the silicon wafer in a clean beaker. Wash the silicon wafer with diluted dish detergent
in an ultrasonic cleaner for 12 minutes.
3. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with ddH2O in an
ultrasonic cleaner for 12 minutes. Repeat this step three times.
4. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with ethanol in an
ultrasonic cleaner for 12 minutes.
5. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with acetone in an
ultrasonic cleaner for 12 minutes.
6. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with ddH2O in an
ultrasonic cleaner for 12 minutes.
7. Transfer it to a clean Petri dish. Dry it in an oven.
8. Drop 30 µL of nanoparticles sample on a silicon wafer.
9. Put it in a vacuum dryer for 1-2 hours, confirm the sample is completely dry.
10. Drop 30 µL of nanoparticles sample on a silicon wafer.
11. Put it in a vacuum dryer for 1-2 hours, confirm the sample is completely dry.
Analysis
1. Using High resolution X-ray Photoelectron Spectrometer (ULVAC-PHI, PHI Quantera Ⅱ) to analyze the elemental composition and distribution in the sample.
RCA
Using ProtoScript® II Reverse Transcriptase
Equipment
1. Thermocycler
Consumables
1. ssRNA ladder (500-9000 bp) (New England Biolabs)
2. Primer 1 (50 μM)
3. Primer 2 (50 μM) *Note: We adopted two primers which differ in sequence and thus the binding
site on the
circRNA, with the attempt to increase the efficiency of RCA.
4. dNTP (10 μM) (Thermo Fisher Scientific)
5. 5X ProtoScript II Reaction Buffer (New England Biolabs)
6. ProtoScript® II Reverse Transcriptase (200 U/µL) (New England Biolabs)
7. 0.1 M DTT (Thermo Fisher Scientific)
8. RNase Inhibitor (40 U/mL) (New England Biolabs)
9. Sample 1 (circRNA) (~50 ng)
10. Sample 2 (IVT product, linear RNA) (~50 ng)
11. Nuclease-free water (Protech Technology)
Protocol
1. Add the following components. (We employed products generated from circularization and IVT to compare the differences between RCA products of circular and linear RNAs, by observing gel electrophoresis result.)
| Reagents | Amount | Amount |
| Sample | CircRNA (variable) | Linear RNA (variable) |
| Primer 1 | 2 μL | 2 μL |
| Primer 2 | 2 μL | 2 μL |
| dNTP | 1 μL | 1 μL |
| Nuclease-free water | to 10 μL | to 10 μL |
| Total | 10 μL | 10 μL |
2. Denature sample RNA & primers for 5 minutes at 65 °C. Spin briefly and put promptly on ice.
3. Add the following components to the denatured reaction.
| Reagents | Amount |
| 5X ProtoScript 2 RT Reaction Buffer | 4 μL |
| 0.1 M DTT | 2 μL |
| ProtoScript II RT | 1 μL |
| RNase Inhibitor | 0.2 μL |
| Nuclease-free water | to 10 μL |
| Total | 10 μL |
4. Incubate the 20 μL RCA reaction in a thermocycler at the following settings.
| Temperature | Time |
| 42 °C | 60 min |
| 65 °C | 20 min |
| 4 °C | ∞ |
**Note: React at 42 °C for one hour, then inactivate the enzyme at 65 °C for 20 minutes.**
5. Pipet 1 μL ssRNA ladder and 9 μL RNA loading dye in a PCR tube, incubate at 90 °C for 2
minutes, then put it on ice for 1~2 mins.
6. Mix 15 μL RCA product with 3 μL dye.
7. Run gel electrophoresis to confirm the size.
8. The remaining cDNA product should be stored at -20 °C.
Using Induro® Reverse Transcriptase
Equipment
1. Thermocycler
Consumables
1. Primer 1 (50 μM)
2. Primer 2 (50 μM) *Note: We adopted two primers which differ in sequence and thus the binding
site on the
circRNA, with the attempt to increase the efficiency of RCA.
3. dNTP (10 mM) (Thermo Fisher Scientific)
4. Induro® RT Reaction Buffer (New England Biolabs)
5. Induro® Reverse Transcriptase (200,000 U/mL) (New England Biolabs)
6. RNase inhibitor (New England Biolabs)
7. ssRNA ladder (New England Biolabs)
8. RNA loading dye (New England Biolabs)
9. Nuclease-free water (Protech Technology)
10. Sample (circRNA) (~50 ng)
11. Sample (IVT product, linear RNA) (~50 ng)
Protocol
1. Add the following components. (We employ products generated from circularization and IVT to compare the differences between RCA products of circular and linear RNAs, by observing gel electrophoresis result.)
| Reagents | Amount | Amount |
| Sample | CircRNA (variable) | Linear RNA (variable) |
| Primer 1 | 2 μL | 2 μL |
| Primer 2 | 2 μL | 2 μL |
| dNTP | 1 μL | 1 μL |
| Nuclease-free water | to 10 μL | to 10 μL |
| Total | 10 μL | 10 μL |
2. Denature at 65 °C, 5 min and keep on ice for 1~2 mins.
3. Add the following components to the denatured reaction.
| Reagents | Amount |
| 5X ProtoScript 2 RT Reaction Buffer | 4 μL |
| RNase inhibitor | 0.2 μL |
| Induro RT | 1 μL |
| Nuclease-free water | 4.8 μL |
| Total | 10 μL |
4. Incubate at 60 °C, 15 mins, then cool down at 4 °C.
5. Pipet 1 μL ssRNA ladder and 9 μL RNA loading dye into a PCR tube, incubate at 90 °C for 2
minutes, then put it on ice for 1~2 mins.
6. Mix 15 μL RCA product with 3 μL dye.
7. Run agarose gel electrophoresis to check the size of the RCA product.
8. The cDNA product should be stored at -20 °C.
Probe conjugated-AuNP detection
Equipment
1. SPECTROstar Nano (UV/Vis spectrometer)
2. Camera (smartphone)
3. Cuvettes
Consumables
1. 0.21 M MgCl2
2. RCA solution with no target
3. RCA solution with target (circRNA)
Protocol
RCA solution with no target
1. Dilute probe conjugated-AuNP solution O.D. to 2.56. Conduct UV-Vis spectroscopy by SPECTROstar
Nano.
2. Pipet 180 µL of probe conjugated-AuNP solution into a cuvette. Add 20 µL of RCA with NTC (no
template control) solution. Use SPECTROstar Nano to conduct UV-Vis spectroscopy to compare the
probe-conjugated AuNP solution with the one without the addition of RCA solution (with no
target). Confirm that there was no redshift in the spectrum or aggregation of the gold
nanoparticles.
3. Add 1 µL 0.21 M MgCl2, react for 2 minutes during each interval. Take a photo and
conduct
UV-Vis spectroscopy by SPECTROstar Nano.
4. Repeat step 3 for multiple times until the solution undergoes a noticeable color change
and a
redshift in the spectrum. Record the volume of MgCl2 added.
RCA solution with target
1. Dilute probe conjugated-AuNP solution O.D. to 2.56. Conduct UV-Vis spectroscopy by SPECTROstar
Nano.
2. Pipet 180 µL probe conjugated-AuNP solution to a cuvette. Add 20 µL RCA solution containing
RCA amplicons. Use SPECTROstar Nano to conduct UV-Vis spectroscopy to compare the probe
conjugated-AuNP solution with the one without addition of RCA solution. Confirm that there was
no redshift in the spectrum or aggregation of the gold nanoparticles.
3. Add the same volume of 0.21 M MgCl2 in the "RCA solution with no target" experiment.
Let it
react for 2 minutes. Take a photo and conduct UV-Vis spectroscopy by SPECTROstar Nano.
4. Confirm the target exists or not by observing the color change after adding nanoparticles
and
MgCl2 to the RCA solution.
Repeated insert sequence synthesis
Insert digestion
Equipment
1. Dry bath
Consumables
1. 10X tangle buffer (Thermo Fisher Scientific)
2. Restriction enzyme: BamH1 (10 U/µL) (Thermo Fisher Scientific)
3. Restriction enzyme: HindIII (10 U/µL) (Thermo Fisher Scientific)
4. Nuclease free water (Protech Technology)
5. Sample (insert)
6. Gel/PCR DNA Isolation System (VIOGENE)
Protocol
1. Preheat dry bath to 37 °C
2. Set up the digestion reaction on ice. Vortex briefly and spin down for 3-5 s.
| Reagents | Amount |
| 10X tangle Buffer | 10 μL (2X ends) |
| Sample | ~1 μg |
| BamH1 | 1 μL |
| HindIII | 2 μL |
| Nuclease-free water | to 50 μL |
| Total | 50 μL |
3. Incubate the digestion mixture at 37 °C for 1 hr.
4. Run the digested DNA on a 1.5 % agarose gel.
5. Conduct gel purification using Gel/PCR DNA Isolation System followed the provided
protocol.
6. Measure a DNA concentration of the purified product by NanoDrop. (About 50 ng is needed for
the following procedure)
7. Use the purified product directly for ligation.
Insert ligation
Equipment
NA
Consumables
1. Digestion product
2. T4 DNA ligase buffer (10X) (New England Biolabs)
3. T4 DNA ligase (New England Biolabs)
4. Sample (Insert)
5. Gel/PCR DNA Isolation System (VIOGENE)
Protocol
1. Set up the ligation reaction on ice. Vortex briefly and spin down for 3-5 s.
| Reagents | Amount |
| T4 DNA ligase buffer (10X) | 2 μL (1X ends) |
| Sample | ~50 ng |
| T4 DNA ligase | 1 μL |
| Nuclease-free water | to 20 μL |
| Total | 20 μL |
2.Incubate at room temperature (25 °C) overnight.
Notice: Since the desired product is about 3000 bp, the incubation time needs to be prolonged
(overnight) for better yields.
3. Run the ligation product on a 1.2 % agarose gel.
4. Conduct gel purification using Gel/PCR DNA Isolation System followed the provided
protocol.
5. Measure a DNA concentration of the purified product by NanoDrop spectrophotometer.
6. Store the insert ligation product at 4 °C.
Preparation of template for AELA-PCR
Ligation with vector
Vector Digestion
Equipment
1. Dry bath
Consumables
1. 10X tangle buffer (Thermo Fisher Scientific)
2. Restriction enzyme: BamH1 (10 U/µL) (Thermo Fisher Scientific)
3. Restriction enzyme: HindIII (10 U/µL) (Thermo Fisher Scientific)
4. Nuclease-free water (Protech Technology)
5. pUC19 vector (New England Biolabs)
Protocol
1. Preheat dry bath to 37 °C
2. Set up the digestion reaction on ice. Vortex briefly and spin down for 3-5 s.
| Reagents | Amount |
| 10X tangle buffer | 10 μL (ends in 2X) |
| pUC19 vector | 0.04 pmol |
| BamH1 | 1 μL |
| HindIII | 2 μL |
| Nuclease-free water | to 50 μL |
| Total | 50 μL |
3. Incubate the digestion mixture at 37 °C for 1 hr.
4. Use the digest vector directly for ligation.
Vector ligation
Equipment
NA
Consumables
1. Vector digestion product
2. Sample (insert ligation product)
3. T4 DNA ligase buffer (10X) (New England Biolabs)
4. T4 DNA ligase (New England Biolabs)
5. Gel/PCR DNA Isolation System (VIOGENE)
6. Nuclease-free water (Protech Technology)
Protocol
1. Set up the ligation reaction on ice. Vortex briefly and spin down for 3-5 s.
| Reagents | Amount |
| 10X T4 DNA ligase buffer | 2 μL (2X ends) |
| Vector digestion product | 0.04 pmol ends |
| Insert ligation product | 0.04 pmol ends |
| T4 DNA ligase | 1 μL |
| Nuclease-free water | to 20 μL |
| Total | 20 μL |
2. Incubate at room temperature (25 °C) overnight.
3. Store the insert ligation product at 4 °C or directly use for transformation.
Transformation
Equipment
1. Dry bath
2. Petri dish
3. Disposable cell spreaders
4. Incubator
Consumables
1. DH5-alpha E. coli
2. Sample (ligation with vector product)
3. LB Medium plate with ampicillin (0.1 mg ampicillin / 1 mL LB)
Protocol
1. Preheat the dry bath to 42 °C.
2. Add 5 μL ligation product into 50 μL DH5-alpha E.coli stock. Do not pipet or vortex, flick
the tube slightly about 5 times.
3. Put the reaction on ice for 2 min.
4. Incubate at 42 °C for precisely 30 sec.
5. Put on ice for 2 min.
6. Add 950 μL cell culture medium.
7. Take 100 μL of the mixture and spread it evenly on a plate using a Disposable cell
spreaders.
8. Incubate the plate in the incubator at 30 °C for 24 hr.
Colony PCR
Equipment
1. Culture tube
2. PCR tube
3. Thermocycler
Consumables
1. LB medium with ampicillin (0.1 mg ampicillin / 1 mL LB)
2. 2X Master Mix (Protech Technology)
3. PCRforAELA_fw primer, 10 μM
4. PCRforAELA_rv primer, 10 μM
5. Sample (transform product)
6. Gel extraction system
Protocol
1. Use pipette tips to pick colony, each Petri dish choose 2 colonies
2. Put the colony in 200 μL of LB with ampicillin (1 μL ampicillin/1mL LB)
3. Shake in a 37°C incubator for 2 hours in preparation for colony PCR.
4. Add the following reagent to PCR tube
| Reagents | Amount |
| 2X Master Mix | 10 μL (1X ends) |
| Free water | 7 μL |
| Foward primer | 0.5 μL |
| Reverse primer | 0.5 μL |
| Sample | 2 μL |
| Total | 20 μL |
5. Conduct PCR at following thermal cycle.
| Stage | Temperature | Time | |
| Denaturation | 94 °C | 1 min | |
| Denaturation | 94 °C | 30 sec | 35 cycles |
| Annealing | 61 °C | 30 sec | |
| Extension | 72 °C | 1 min 5 sec | |
| Extension | 72 °C | 7 min | |
| Cooling | 4 °C | ∞ |
6. Run the PCR product on a 1.2 % Agarose Gel.
7. We found out that there was no band presented on the agarose gel, we want to purify the
plasmid to run gel electrophoresis instead. To extract plasmid, transfer 10 μL bacteria liquid
to 5 mL LB with ampicillin in a culture tube first.
8. Shake in a 37 °C incubator for 16 hours.
Plasmid extraction
Equipment
1. Centrifuge tube
Consumables
1. Mini Plus Plasmid DNA Extraction System (Viogene)
Protocol
1. Perform plasmid extraction follow the protocol provided by the manufacturer.
2. Run the extracted plasmid on a 1.2 % agarose gel to identify whether the ligation process was
successful.
AELA-PCR
Equipment
1. PCR tubes
2. Thermocycler
Consumables
1. i-Taq 2X PCR master mix kit (Intron Biotechnologies)
(the kit contains 2.5 U/μL of DNA polymerase, 2.5 mM each of dNTPs, and 1X reaction buffer with
3 mM MgCl2)
2. Forward primer (0.2 μM)
3. Reverse primer (10 μM) *Note: The concentration ratio of the extended (rv) primer to
un-extended (fw) primer should be 20:1. (See Parts_AELA_PCR)
4. Nuclease-free water (Protech Technology)
5. Sample (long-repeated dsDNA)
Protocol
1. Add the following reagents to PCR tube.
| Reagents | Amount |
| i-Taq mix | 10 μL |
| Primer mix (extended primer:un-extended primer = 20:1) | 1 μL |
| Long-repeated dsDNA sample | 1 μL |
| Nuclease-free water | 8 μL |
| Total | 20 μL |
2. Conduct PCR at following thermal cycle.
3. React in a thermocycler at following setting.
| Stage | Temperature | Time | |
| Denaturation of dsDNA | 95 °C | 2 min | |
| Exponential phase | 95 °C | 20 sec | 35 cycles |
| 58 °C | 15 sec | ||
| 72 °C | 40 sec | ||
| Linear phase | 95 °C | 20 sec | 25 cycles |
| 72 °C | 50 sec | ||
| Forming ssDNA amplicons | 72 °C | 2 min |
4. Conduct agarose gel electrophoresis to confirm our PCR products.
RPA
Equipment
1. 8 Low Profile PCR tube
2. Thermocycler
3. Mini centrifuge
Consumables
1. Rehydration buffer (TwistDx)
2. Forward primer (10 µM)
3. Reverse primer (10 µM)
4. MgOAc (280 mM) (TwistDx)
5. RNAse-free water (Protech Technology)
6. Sample (insert)
7. TwistAmp® Basic reaction pellets tubes (TwistDx)
Protocol
1. Add the following reagents to TwistAmp® Basic reaction pellets tubes.
| Reagents | Amount |
| Primer free rehydration buffer | 29.5 μL |
| Foward primer | 2.4 μL |
| Reverse primer | 2.4 μL |
| Sample | ~100 ng |
| RNase-free water | to 47.5 μL |
| Total | 47.5 μL |
**Note: Forward primer and reverse primer are designed by ourselves. For more details, please view Parts_RPA primer.**
2. Add 2.5 µL of 280 mM MgOAc to the lid of TwistAmp® Basic reaction pellets tubes.
3. Spin down with a mini centrifuge to introduce MgOAc into the rehydrated material and initiate
the reactions.
4. Vortex briefly and spin down once again.
5. Incubate the tubes in thermocycler under 37˚C for 20 minutes.
6. After 20 minutes, run gel electrophoresis to confirm if the amplification is successful. For
the lateral flow test, follow the prescribed instructions to appropriately dilute the reaction
products.
RT-RPA
Equipment
1. Thermocycler
Consumables
1. Primer free rehydration buffer (TwistDx)
2. Forward primer (10 µM)
3. Reverse primer (10 µM)
4. ProtoScript® II Reverse Transcriptase (200 U/µL) (New England Biolabs)
5. RNase inhibitor (New England Biolabs)
6. dNTP (10 mM) (Thermo Fischer Scientific)
7. RNase free water (Protech Technology)
8. Sample (IVT product, linear RNA)
Protocol
1. Prepare reaction mix in 1.5 mL tube
| Reagents | Amount |
| Primer free rehydration buffer | 29.5 μL |
| Foward primer | 2.4 μL |
| Reverse primer | 2.4 μL |
| Protoscript RT | 0.5 μL |
| RNase inhibitor | 0.5 μL |
| dNTP | 1 μL |
| Sample | 2 μL |
| RNase-free water | to 50 μL |
| Total | 50 μL |
2. Add reaction mix to a TwistAmp® Basic reaction. Pipette to mix.
3. Add 2.5 μL of 280 mM Magnesium Acetate (MgOAc) and mix well to start the reaction. *Note: RPA
reactions start as soon as MgOAc is added.
4. Incubate at 39 ˚C for 20 minutes.
5. After 20 minutes, run gel electrophoresis to confirm if the amplification is successful. For
the lateral flow test, follow the prescribed instructions to appropriately dilute the reaction
products.
PCRD
Equipment
NA
Consumables
1. PCRD (lateral flow strip cassette) (Abingdon Health)
2. PCRD Extraction Buffer (Abingdon Health)
3. Sample (RT-RPA product)
Protocol
1. Pipette 6 µL of the amplification product into a 0.5 mL tube.
2. Transfer 84 µL of the PCRD Extraction Buffer supplied into the tube and mix thoroughly.
3. Open the foiled PCRD, add 75 µL of the diluted reaction mixture to the sample well of a PCRD
test cassette.
4. Leave the cassette in a horizontal position for 10 mins while the result develops. Read the
result visually at 10 mins. Ignore any changes which occur after 10 mins.