Overview
The workflow of our experiments is based on the design of our project, which consists of three parts : HCR + CRISPR verification, HCR system optimization, and paper-based hardware verification . In this page, you can find the experimental program used during the experiment, detailed experimental design.
The most critical step in laboratory work is to report the daily experiments in detail. The following is our team 's experimental book, describing our experimental procedures. To download it, click “Lab Book” to get our experimental log.
Experiments
The following is the main design of each part of the experiment and the corresponding documentation links.
Feasibility validation
1.HCR verification
Firstly, in order to verify the feasibility of HCR-CRISPR detection of miRNA, we designed the following experiments, and verified the results by gel electrophoresis and fluorescence detection to confirm the feasibility of the detection. The main design of this part is shown in Table.1.
To download our complete experiment, click the box below.
2.Feasibility verification of HCR + CRIPSR
The main design of this part is shown in Table.2.
To download our complete experiment, click the box below.
3.Determination of specificity, detection limit and sensitivity
After that, we verified the specificity of our system, and determined the detection limit and sensitivity. The following shows only specific experimental designs, as shown in Table. 3.
To download our complete experimental protocol for the determination of specificity, detection limit, and sensitivity, click the corresponding box below.
Optimization
In order to screen better reaction conditions and provide a better environment for the HCR + CRISPR reaction, we optimized several aspects of the experimental system ( including H1 stem-loop length, H1 probe ratio, Na + ion concentration, etc. )
The following shows an optimized experimental design for stem-loop length only for H1-98-5p, as shown in Table.4.
To obtain our complete experimental scheme to see more optimized designs, click the corresponding box below.
Hardware: Paper-based verification
1.Pre-experiment part (compatibility test)
In order to test the compatibility of hardware and biological reaction, we first carried out HCR reaction experiments in solution systems with different carriers, including HCR reaction in pure biological process ( laboratory environment ), HCR reaction on reaction pad in solution system, and HCR reaction on paper-based chip in solution system.
The following shows only the HCR reaction on the reaction pad in the solution system, as shown in Table.5.
To download our hardware-compatibility test complete experimental program, click the corresponding box below.
2.Drying test (tolerance test)
Next, we performed a tolerance test, that is, reacting on a dry reaction pad. The following only shows the HCR reaction on the dry reaction pad, as shown in Table.6.
To download the complete experimental scheme of our hardware-tolerance test, click the corresponding box below.
3.HCR + CRISPR reaction on paper-based chips
On a paper-based chip, we verified HCR + CRISPR. It has been verified that the paper-based chip can occur normally and output fluorescence normally. To download the complete experimental scheme of our hardware-tolerance test, click the corresponding box below.