Meeting Gold Medal Criteria
- We have designed accessible extraction and fluorescence detection kits, posing value in field testing, and reducing the costs of research.
- We have attached 3D models (of the hand centrifuge, micropestle, dark box and testing tray) in several formats to make further adaptation and use as accessible as possible.
- We have included a descriptive protocol, including videos, for the extraction protocol we designed.
- For the dark box PCB, a commented schematic and layout have been included.
- The PCB was made for simple hand solder assembly.
- The smartphone app is fully documented, using cross-platform libraries to be easily developed and tested on multiple platforms, and includes shell scripts to simplify code generation.
- We conducted statistical tests on the detection device to confirm the accuracy of the fluorescent readout. We also used several techniques to compare the extraction protocols to lab standard procedures.
Introduction:
Throughout this project we have developed several hardware tools to aid in the extraction and detection of RNA in plant samples. This posed a significant challenge, as RNA is inherently unstable, and many conventional extraction methods depend on expensive equipment (centrifuges and micropipettes) as well as technical expertise. We designed and optimised an extraction kit and protocol with accessibility in mind, with the goal of circumventing the issues faced by those without access to well equipped labs - such as smallholder farmers.
The extraction kit includes a hand centrifuge, pestle and ‘Syringe Pipette’. In addition to this, we designed a detection device: a reusable dark box, a tray for loading the samples, and a compatible smartphone app, so that detection, as well as the extraction, is as accessible as possible.
Field extraction kit
The field extraction is an adaptation of the isopropanol RNA extraction, to be made accessible for farmers. This required developing an efficient alternative to lab standard centrifugation, homogenisation and micropipette techniques - hence the hand centrifuge, micropestle and Syringe Pipette.
The full written protocol can be found below:
The Hand Centrifuge
Building from a prior model1 for a hand powered centrifuge, we made several improvements to functionality, with RNA extraction in mind - such as adding cooling capabilities. We also improved the ease of use, developing a more effective mechanism for securing the tubes, relative to the original design. To make sure the test is accessible and easy to follow, we filmed an instructional safety video, detailing how to perform the centrifugation safely.
Testing cooling capabilities
The original centrifuge was very simple, only capable of holding the samples. In order to cool the centrifuge, we needed to add water channels parallel to the PCR tube holder. These could then be filled with water and frozen. We went through two iterations, the first was open, the final design covered the channels.
Results
The original and cooled centrifuges were scaled up to fit a thermometer in, then placed in the freezer (the new design filled with water). The temperature was then recorded over 40 minutes, to determine the relative cooling effect of adding water channels.
While this data shows how effective the water channels are while cooling, we noticed that in reality the ice melted far faster while spinning the centrifuge. To examine the effect of this, and the difference of covering the channels, we froze both iterations, then span each over a span of 14 minutes, weighing them every minute from 2 minutes onwards. We used the same length string for both experiments, and taped over the open water channels.
Both experienced a similar initial rate of cooling, however the covered design retained water for longer (12 minus), relative to the taped design (6 minutes) - indicating a marked improvement on the centrifuge’s capacity to reduce the extent to which samples are heated while spinning.
This improvement to the centrifuge keeps the sample cool while precipitating in the 15 minute spin step, increasing both the quantity, and integrity of the RNA precipitated.
Holding the tubes in
While spinning, unless taped down, PCR tubes would fly out of the centrifuge. While taping would ordinarily pose a viable solution, the frozen centrifuge is slightly damp, and as such, tape does not stick well to the plastic. Our solution to tubes flying out was to develop simple fastening systems:
Micropestle
To aid with homogenisation, and resuspension of the final pellet, we designed a double sided micropestle for PCR tubes. The double sided pestle means that one end can be used for homogenisation, while the other remains relatively clean for resuspension.
Syringe Pipette
The next issue was developing a method which would allow farmers to transfer small volumes of liquid between PCR tubes. As all reagents are premeasured, it was only a question of transferring the entire contents into or out of a tube, so precision was not necessary. Our solution to this was to use robotic pipette tips inside plastic syringes, a 230μL one was long enough to fit through the syringe, while also being narrow enough to fit.
Testing the Extraction
We used several methods to compare the efficacy of the protocol relative to lab standard techniques conducted under RNase free conditions. These included nanodrop, ScreenTape and RT qPCR results.
It is worth noting that for these tests the health of the A.thaliana was very poor, and this may have significantly affected absolute values (though relative comparisons between techniques are still valid). This is reflected in the RNA ScreenTape results, which shows ~½ the concentration for standard isopropanol extraction relative to prior instances following an identical protocol.
Nanodrop results:
| [RNA] ng/μL | 260/280 | 260/230 | |
|---|---|---|---|
| Isopropanol | 101.9 | 1.49 | 0.56 |
| Farmer | 227.6 | 1.32 | 0.57 |
ScreenTape results:
The farmer extraction was repeated twice on the ScreenTape to be efficient with our ScreenTape device as the machine runs samples in pairs.
RT-qPCR results:
Cq values obtained from each detection method, when DIY and TF primers were used, separately. Undetermined Cq values were obtained for some no reverse transcriptase (RT) controls, so no bars were plotted for these wells. WHT: Whatman, ISP: Isopropanol, FRM: Farmer extraction, TRZ: TRIzol extraction.
This demonstrates that the farmer extraction protocol is of a comparable standard to a normal lab isopropanol protocol, as well as other standard lab techniques, despite the less efficient homogenisation process, and working outside of RNase free conditions.
Testing accessibility
This whole protocol was tested by a member of the team who had not used the equipment, or done any other extraction protocols. She was able to extract RNA successfully (confirmed by nanodrop tests (55.8 ng/μL and 260/280 = 1.53). Her main comment was on the need for a PCR tube rack to place tubes on, as such we implemented this into the dark box design. She also suggested some amendments to the protocol to improve clarity (such as preventing contamination of the micropestle).
Detection
In order to make a reliable and sensitive diagnostic tool for detecting fluorescent signals, we designed a dark box and smartphone app. We decided dedicated hardware was necessary over visual inspection, despite cost disadvantages, as it would remove the requirement for skill, make the test standardised, and accessible
We decided that a smart phone based device would provide the best balance between sensitivity, accessibility and cost. With inspiration from our review of the work of previous iGEM teams2, we decided to develop a dark box, to provide a controlled and optimal environment for capturing the fluorescence, alongside an app to actually perform the capture, and any additional post-processing necessary.
The dark box is a 3D printed structure that allows you to place your phone inside, to take pictures of the test samples in the dark. Across multiple iterations, we improved our dark box in a number of ways: we optimised the LED positioning to reduce glare, we improved the box height to best match the focal length of different cameras, and added grooves to precisely control the positioning of each sample disk in the frame of the camera. As mentioned earlier, following feedback from another team member on the extraction protocol, a PCR tube rack was integrated into the final design of the dark box.
The final procedure for using the box involves placing the phone above the hole for the camera. The LED is then inserted into its holder and turned on, illuminating the inside with the correct wavelengths of light for excitation of the ThT fluorophore. The sample tray can then be inserted through the opening into the box. The tray has grooves allowing it to lock into place with the negative control directly below the camera when pushed all the way in. Pulling it out slides the tray until it locks with the positive control below the camera. Again, pulling further locks the test sample below the camera. At all these positions a photo can be taken, for the app to then process and give a readout.
During testing, we used an Arduino Uno with a 405nm LED for exciting the ThT. This worked well for development and prototyping, as the Arduino provided simple programmatic control over the LED. The final product, however, will use a PCB in order to simplify the usage and reduce the cost. The design was guided by a few key criteria - minimising cost, maintaining accessibility for farmers and ease of adaptation for future iGEM projects. To keep costs low, as few parts as possible were used - i.e. not including a switch, as unplugging the device works equally well. Farmer accessibility was also considered, explaining the use of the Micro USB-B plug over others like a USB-B plug. Micro USB-B is also cheaper than potential alternatives like USB-C. To facilitate further development and ease of assembly for future iGEM teams, the design also uses solely through hole mounted components. This makes it easy to hand solder, allowing potential prototyping and self assembly. Unfortunately, due to time constraints, we were unable to get the PCB fabricated and built, but verification using tools present in KiCAD gives some level of confidence to the designs. The bill of materials and documented CAD files are available for download at the bottom of the page.
Alongside the hardware, software was necessary for the detection of the fluorescence. Simply taking an image of the fluorescent disc suffers multiple issues, such as there being no reference for how bright the fluorophore is, or the fact that the LED is too bright, meaning it often drowns out the fluorophore’s fluorescent light. To combat this, we developed an algorithm that takes the pixel-wise difference between the positive test and the negative control. Any difference in pixel values provides what changed between capturing the images. If fluorescence was present, that is then isolated in the output picture.
Detection Testing
After an initial round of testing, we found that the main idea behind the algorithm was successful, however we found it was unreliable due to noise and errors when taking the pictures. This noise often came from light seeping in through cracks from the tray entrance. Therefore, if our program could automatically crop out the majority of the border around the sample, almost all present noise would be removed.
This automatic cropping was achieved by drawing 4 dots around the negative control. These were found using simple blob detection, allowing the negative control image to be cropped to the right size. Thanks to the grooves and locking mechanism, the test sample would move into exactly the same position the negative control was in. This means using the same pixel coordinates lets you crop out the new sample disc without having to reperform the blob detection.The subtraction is then performed and the results were significantly better, especially after also implementing auto-focus.
We compared the ability of the new and old models to discern concentrations of ThT, in both black and white dark boxes:
| SRCC values | Old, n = 7 (Critical Value =0.7143) | New, n = 8 (Critical Value =0.6429) |
|---|---|---|
| Black | 0.5357142857 | 0.8095238095 |
| White | 0.3571428571 | 0.9375872052 |
The improvements to the detection kit show a marked increase in correlation between concentration of ThT and detected brightness, showing how effective these changes are in improving quantification.
We also tested how the entire distribution of differences in pixel brightness changes as we varied the Thioflavin T concentration, in a white and a black environment, to determine which is better for our application. We suspect that the plateau at 50,000 pixels is representative of the size of the sample disc, in the images taken - a rough upper ceiling for the higher brightness values. In this we see the white dark box has a more consistent plateau across several concentrations, indicating that it may be better for discerning the sample disc from the rest of the dark box. However, the black dark box shows a better correlation between brightness and Log10([ThT]) (PMCC test, r = 0.9632902513 vs 0.9517549052, n=7, p<0.05). Both show strong correlations, however the black dark box shows more promise at lower concentrations and fluorescence intensities - indicating it may be more sensitive. Both designs have different strengths, and demonstrate potential for detecting quantitative readouts, given a positive control to calibrate with.
Overall, the app and box show a very promising foundational tool for cheap, paper based detection of fluorescent tests. Having shared all of our resources for this, they can also act as a platform for future researchers and iGEM teams to build off of and extend with new capabilities.
Cost estimation
Price per test:
These expenses were calculated using retail prices. In reality a test would likely be cheaper, when purchasing at an industrial level, and potentially manufacturing our own enzymes, such as phi29 DNA polymerase.
Extraction Pricing
RCA pricing
Hybridisation pricing
| Item | Price | Price per Unit | Source |
|---|---|---|---|
| Whatman 1 paper discs | £197/100 sheets (each sheet gives ~9000 discs) | £0.00022 | https://www.fishersci.co.uk/shop/products/whatman-qualitative-filter-paper-grade-1-sheets/11310404 |
| APTMS | £287/500ml | £0.0000025 | https://www.sigmaaldrich.com/GB/en/product/aldrich/281778?srsltid=AfmBOoqj9SjSKL-9VYBRp0SQ25XsbgVrDQr3_4PZYY63vuVwBO-ypoKw |
| EtOH (~5mL) | £91.50/25L | £0.018 | https://apcpure.com/product/denatured-ethanol-995/GPS9065-F?gbraid=0AAAAADk1sy4nzJRZ7fjoawkpel0jFHqq2 |
| Immobilised DNA probe (2uL 10uM) | £2.74/100nmol | £0.00055 | IDT |
| PBS | £1430/50kg | ||
| Mobile DNA probe | £60.36/100nmol | £0.0097 | IDT |
| 20xSSC (~0.5mL) | £35.50/1L | £0.018 | https://www.sigmaaldrich.com/GB/en/product/sigma/s6639?srsltid=AfmBOoraad_b8YAvk-SFKbfOGFtI7EPIG80U3flow9P5nopMOl8VtbE- |
| SDS (~0.001g) | Negligible | ||
| Tween-20 | Negligible | ||
| BSA | Negligible | ||
| DI water | Negligible |
Total per unit: £7.57+ (3.75*4 RCA paper discs or £0.05*3 hybridisation paper discs) = £22.57 or £7.72
One off Expenses:
Total one off costs: £6.21
Further Applications
While designing an RNA diagnostic test for farmers, we also developed a viable and accessible extraction protocol, with the farmers in mind. We hope that hardware from this, such as the 3D printed centrifuge, can be put to use elsewhere, perhaps in field test kits for different protocols. We also hope that the detection hardware (and corresponding app), can be put to use by other iGEM teams and researchers in paper based and/or fluorescent diagnostics. Hopefully this improves the overall accessibility of research providing cheaper alternatives to expensive and technical processes.
Conclusion
We have designed a simple and efficient hardware kit to facilitate accessible extraction and detection of RNA. This is accompanied by written and video protocols, as well as a compatible smartphone app. The kit has been tested and optimised for RNA extraction - however we hope that the parts will be used in new and creative ways, by other iGEM teams.
We have provided 3D models for all printed parts in STL, STEP and PARASOLID formats, in the hope of making them as accessible as possible
hardware files - files for 3D models in several formats
The app code can be found at our software tools repo.
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