OVERVIEW
To test our modules of biomarker detection, we developed various experimental methods. For each module, we had the following:
I) For the miRNA detection module:
FASTmiR is a suitable tracking method for the detection of miRNAs.
- PCR Amplification of the sequences: To amplify the number of sequences and check their lengths, we decided to first optimise PCR conditions by setting up a Test PCR. After its standardisation, we would increase the reaction volume and set up a scale-up PCR.
- In-Vitro Transcription: As our desired sequences were RNA, we would have to conduct an in-vitro transcription to fetch the RNA sequence.
- Incubation: We had to check the fluorescence intensity through various DFHBI, RNA and DNA concentrations in different incubation periods.
- Fluoroscence Measurement:
After incubation, we would finally measure the fluorescence emitted using a plate reader.
Overview of the Experimental workflow:
II) For the protein and hormone detection module:
The dual-aptamer technique works on the proper functioning of three sequences. The technique works when an annealed complex of aptamer with a trigger is bounded by a biomarker. When another sequence (the target) with a higher complementarity than the aptamer-trigger complex is near it due to conformational change by the biomarker binding, the trigger sequence gets released and forms another complex; the trigger-target sequence. The biomarker also creates a conformational change in the aptamer to form a dye-binding pocket, to which DFHBI binds and produces fluorescence.
Hence to prove the functionality of this system, we divided our experimental approach into the following parts:
- To prove the annealing of aptamer-trigger sequences: By comparing molecular weights and length between only aptamer control, only trigger control and aptamer-trigger control after incubation in a native PAGE.
- To prove the annealing of trigger-target sequence: By comparing molecular weights and length between trigger control, target control and trigger-target control after incubation in a native PAGE.
- To prove the release of the trigger after incubating with the biomarker: By detecting double bands, which would denote the splitting of the aptamer-trigger complex, where one band denotes the aptamer and another band denotes the trigger, respectively.
Overview of the Experimental workflow:
