Application of cdr1as in prenatal screening for neural tube defects, products and methods for prenatal screening for neural tube defects
A neural tube defect and prenatal screening technology, applied in the field of clinical diagnosis, can solve problems such as inability to diagnose, and achieve the effects of low professional requirements, wide universality, and sensitive and fast detection cost.
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Embodiment 1
[0051] Example 1 Screening of Markers
[0052] Neural tube tissue samples from 10 groups of children with NTDs (experimental samples) and 10 groups of normal fresh neural tube tissue samples (control samples) of similar age were collected. The circular RNA chip was sequenced, and microarray analysis was performed to select the five circular RNAs with the largest fold difference, including circRNA-CDR1as, Circ-TTBL C2, Circ-MYLK, CZNF292 and CircRNA100876.
[0053] For the test samples and control samples, the extracted total RNA was reverse-transcribed with random primers of the reverse transcription kit, circRNA-CDR1as primers were designed, and the expression levels of CDR1as in the test samples and control samples were detected by RT-PCR. The extent to which CDR1as was elevated in the test samples. The result is as figure 1 Among them, the expression difference of circRNA-CDR1as was the most significant.
[0054] Among them, the circRNA-CDR1as primers are as follows:
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Embodiment 2
[0059] Example 2 The effect of circRNA-CDR1as on cell proliferation
[0060]Plasmids expressing circRNA-CDR1as were constructed using pcDNA3.1, embryonic stem cells were transiently transfected with lipo2000, and then induced with 5uM RA to successfully induce differentiation into neural stem cells to construct neural stem cells overexpressing circRNA-CDR1as. The neural stem cells after induction and differentiation were subjected to the following experiments: incubate the neural stem cells with CCK8, detect the absorbance at 450 nm of the cells on the 1st, 2nd, 3rd, 4th, 5th, and 6th days, respectively, and do the cell proliferation and growth curves to reflect the proliferation. The result is as figure 2 As shown, neural stem cells overexpressed by circRNA-CDR1as proliferated faster than those in which pcDNA3.1 plasmid was empty, indicating that circRNA-CDR1as could promote cell proliferation.
[0061] The siRNA of circRNA-CDR1as was designed and purchased from Ribo Biotec...
Embodiment 3
[0063] Example 3 The effect of circRNA-CDR1as on the expression level of TRIM-71
[0064] Referring to the experiment in Example 2, circRNA-CDR1as-overexpressing neural stem cells and idling pcDNA3.1 plasmid neural stem cells were obtained, and the above two cells were cultured with or without cycloheximide (Dactinomycin) medium, respectively. The total protein in the cells was extracted by the whole protein extraction kit. After the protein concentration was determined by the BCA protein concentration assay kit, each group was diluted to the same concentration and added to the loading buffer for denaturation, and detected by western blot. In the experiment, GAPDH was used as the internal reference. The differences in the expression of TRIM-71 in cells were compared. The result is as Figure 4 As shown, the protein amount of TRIM71 in neural stem cells overexpressed by circRNA-CDR1as is less than that in neural stem cells with empty pcDNA3.1 plasmid. expression.
[0065] Re...
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