Universal hairpin primer and application thereof in detection of microRNA
A hairpin and reverse primer technology, applied in the field of molecular biology, can solve the problems of low cost efficiency, low detection efficiency of general stem-loop primers, time-consuming and labor-intensive problems, achieve low cost, reduce production cost and detection cost, and simple method convenient effect
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Embodiment 1
[0040] Embodiment 1. A method for detecting microRNA based on universal hairpin primers, the steps are as follows.
[0041] 1) Cell culture: Culture the cells to be tested in conventional cell culture media such as DMEM or F12 containing 10% fetal bovine serum, 100 U / ml penicillin and 100 µg / ml streptomycin, placed at 37°C, 5% CO 2 In an incubator, culture until the cell confluency is above 90%. Different treatments were given according to the requirements of the experiment, and the end point of the experiment was detected.
[0042] 2) Total RNA isolation and small RNA purification: Use NucleoZOL RNA Isolation Kit (Takara Bio) to isolate total RNA from each group of exponentially growing cells to be tested. 5 µg of total RNA was dissolved in 20 µl of RNase-free ddHO and mixed with 20 µl of Mag Bind® TotalPure NGS magnetic beads (Omega Bio-tek, RNA:beads 1:1 v / v ratio). The RNA / bead mixture was incubated at room temperature for 10 minutes. Place the mixture on a magnet, col...
Embodiment 2
[0049] Example 2. Evaluation of the effectiveness of detecting microRNA with different universal hairpin primers
[0050] The cells to be tested are human embryonic kidney HEK-293 cells, human osteosarcoma 143B cells and human melanoma A375. The qPCR template was obtained according to the steps in Example 1, and the template was subjected to five-fold serial dilution to determine the amplification efficiency of each qPCR primer pair. No template control (NTC) was used as a negative control. To quantitatively evaluate the Cq deviation from the microRNA-specific hairpin primer (MsHP) group, ΔCq of the UHP group = mean Cq (MsHP) - mean Cq (UHP). All qPCR reactions were set up in triplicate, and three independent batches of experiments were performed.
[0051] Data analysis: Linear mixed-effects models were fitted by restricted maximum likelihood and compared with the Cq values generated by MsHP to determine the most appropriate UHP. A nonparametric Kruskal-Wallis test was us...
Embodiment 3
[0056] Embodiment 3. The preferred scheme of UHP composition:
[0057] UHP2, UHP4 and / or UHP6 were mixed with different mole percentage compositions to obtain 15 UHP compositions, namely Mix1 to Mix15 (see Table 1). Based on the above 15 UHPs compositions for microRNA detection, each Cq value of 14 microRNAs was obtained and compared with the Cq value of the corresponding MsHP.
[0058] Table 1. Mole percent of each component in 15 UHP compositions
[0059]
[0060] Heatmap clustering analysis of the Cq values of the four tested microRNAs showed that Mix3 clustered with MsHP, while Mix4 and Mix12 clustered closely with UHP4. Among all 15 groups, the Mix3 group had the least deviation relative to the MsHPs group, while most other groups including UHP4 tended to have lower Cq values than the MsHPs group, that is, overestimated microRNA expression levels. Statistical differences in Cq values between Mix3 and UHP4 were shown by boxplot analysis (see Figure 6 ), indica...
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