Team:NUDT CHINA/Demonstrate
PAGE STILL UNDER CONSTRUCTION
Demonstration
FOR FURTHER DEMONSTRATION OF OUR PROJECT, let-7a was dissolved in 7% mixed human serum collected from 50 healthy volunteers on various concentrations to assess the reliability, sensibility and specificity of our scheme.
Similarly, the sensibility and specificity of RCA reaction were then determined with sybr I fluorescence assay (Figure 1). MiR let-7a, as the target miRNA was DISOLVED IN 7% HUMAN SERUM for various concentrations to determine the sensitivity of RCA reaction (Figure 1A). The best reaction time under such circumstance was determined to be 150min through a Real-time fluorescent assay using Sybr I as the Fluorescent dye indicating the dsDNA amount in reaction solution (Figure 1B and C). The sensitivity of such system was estimated to be under 1 fM building on a plotting the ΔFI data against minus logarithm of let-7a concentration and its fitting curve (Figure 1D). A brilliant specificity of RCA reaction was also shown by evaluating the RCA reaction strength under the input miRNA of let-7a, let-7c, let-7f and let-7g (Figure 1E).
(Figure 1)
Subsequently, RCA outputs were then used for dCas9 binding and down-stream HRP activity analysis (Figure 2A). Through which, the preciously-determined protein concentration was further validated and a basic demonstration of our project was presented (Figure 2B and C).
(Figure2)
MOREOVER, as a deepened demonstration of the whole detection workflow, serum samples collected from NSCLC patients and healthy volunteers were tested after different pretreatment for further demonstration of our scheme. For such matters, we collected serum samples from 5 healthy volunteers and 5 volunteered non-small cell lung cancer (NSCLC) patients under their consent. Patient group was formed by two phase III patients, two phase IV patients and one phase V patient. To evaluate the optimal explosion effect of the serum miRNAs, which were previously reported to mainly exist as RNA-protein complex or even in exosomes, for further detection, we used different methods for the pre-treatment of serum samples (Figure 3A). RCA reaction followed by a Sybr I fluorescence assay revealed that both the approaches of serum RNA extraction by TRIzol LS and 10% serum boiled in 95℃ for 15 min can show a significantly lower concentration of let-7a in NSCLC patients compared to healthy people, and the latter is more convenient and effective (Figure 3B). Which then, made the low-cost, high efficient RNA extraction possible for field-ready detection of miRNAs in serum sample. The concentration difference of serum let-7a between NSCLC patients and healthy people determined by our method was corroborated to the previous literature reported 20%-60% decrease measured through qRT-PCR1.
(Figure3)
Generally speaking, brilliant specificity and sensitivity was achieved in stimulated clinical condition and actual patient samples. Thus provided a solid demonstration of our scheme and a sound support for the further development of such system.
The detailed results could be found on the RESULT PAGE.
1 Jeong, H. C. et al. Aberrant expression of let-7a miRNA in the blood of non-small cell lung cancer patients. Mol Med Rep 4, 383-387, doi:10.3892/mmr.2011.430 (2011).
