Team:SZTA RMG Szeged/Implementation
Implementation
Contents
I. Introduction
- We have read about miRNAs and what functions they serve in cancer detection.
- MicroRNAs are small, highly conserved non-coding RNA molecules involved in the regulation of gene expression.
- „Recently, numerous studies demonstrated that microRNAs are emerging as diagnostic biomarkers for (for example kidney) cancer. Specific miRNA profiles have been identified for several samples from patients with kidney cancer.” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5419229/
II. Our project
- We were thinking about that the miRNAs statement could provide huge help in early diagnosing in large quantities for those people who are suffering in cancer; and how we can detect it faster and cheaper than the current methods.
- Our project is mainly aiming for tests that don’t need expensive machines, in addition it is cheap, fast and environmentally friendly.
- With this detection process it would be easier to get the right treatment at a Polyclinic or Public Clinics. For the General Practitioners it can also be a more convenient way to diagnose patients.
- Our project primarily targets physicians, emergency physicians, pathologists and indirectly patients as a social target group.
1. We used ssDNA molecules as templates both for the second strand DNA and
the RNA synthesis.
2. We attached the ssDNA molecules to magnetic beads (Dynabeads®) by their
3’-end with a Biotin-Streptavidin linkage. It was necessary to avoid
a. the effect of 3’à5’ DNAse activity of the chosen DNA polymerases
b. the attachment of the different ssDNAs we used in the same reaction
mixture.
3. First, the sample RNA (saRNA), that is shorter than the single-stranded DNA
binds to the appropriate ssDNA sequence part.
4. We have to use a DNA polymerase which accepts RNA as a primer. Therefore,
we’ve chosen the large Klenow fragment and the large fragment of Bsm polymerase.
5. Then the chosen DNA polymerase enzyme synthesizes a dsDNA, making the
fully double-stranded DNA.
6. After this, the T7 polymerase is attached to the redesigned T7 promoter, that
synthetizes multiple single-stranded RNA (ssRNA) from the DNA that had already
been created by the DNA polymerase. The T7 polymerase accepts its promoter
exclusively if it is double stranded. This feature ensures that the forthcoming
transcription step cannot start until the previous DNA synthesis has not finished.
7. The transcription process is terminated when T7 RNAP reaches the attached
saRNA, as it can’t be used as an antisense strand.
8. The transcribed RNA has a sequence which can hybridize to the next similar
ssDNA.
9. The whole cycle is repeated three more times producing a large quantity of
RNA and dsDNA.
10. The amount of the amplified nucleic acids can be assessed in two ways:
-Thanks to the EcoRV restriction site we may cut the dsDNA molecules and
separate them from the beads. The dsDNA concentration can be characterized by
an intercalating dye (such as SYBR Green I).
- The other, more simple method is the detection of the amount of the
amplified RNA (e.g.: by SYBR Green II). (for more details watch our video)
- One of the biggest advantages in our project is it doesn’t need any huge and expensive devices. The currently used methods need a Real-time PCR machine that costs 20,000-30,000 USD. Despite that we use a spectrophotometer to get the results that costs 600 USD. We detected the amplified RNAs by SYBR Green II dyes. We place the prepared samples into a PCR tube holder stand that was printed out with a three- dimensional printer. In this way, we can measure the fluorescence of a hundred times larger amount of samples than if we measured in cuvettes.
- To get done the test we need further devices that belong to basic equipments of a laboratory. We can recycle the magnetic beads to make our project more environmentally friendly. We incubated the used magnetic beads at 95 °C for 30 minutes, since research suggests that at that temperature the beads are no longer able to immobilize DNA. This step allows us to reuse the beads multiple times to detect RNA, so it significantly decreases the price of our method, given that the magnetic beads are the most expensive component of the detection.
- Proposed solution:
| - | In our Project | Current methods |
|---|---|---|
| Real-time PCR machine | - | + |
| Chemicals | + | + |
| Reusable chemicals | + | - |
III. Safety
- In order to ensure the validity of the test we need some safety aspects. A body fluid sample is collected from the patient and we need to keep them in a cold, sealed, RNAse-free environment. The biggest difficulty of our project is to sustain these conditions, especially while used in less developed surroundings.
IV. Challenges
- When using the test, we must also take into account that the higher concentration of the selected biomarker is likely not unique to type of cancer only. The results may be unreliable in the case of pregnant women, since pregnancy causes the miRNA levels to elevate in the body.
- Another problem is that other components of body fluid can become falsely tinted, to minimize the risk of this and get more accurate results we must first purify the miRNA from the sample.
V. Future use
- Our tool can basically detect any selected RNA. We even made a software that generates the sequence of the needed ssDNA probes to detect any given RNA, to facilitate the process. This way the method is more accessible to other scientists for other implementations.
