Kit for detecting double-stranded RNA of RNAi-based transgenic plants and application of kit
A technology of transgenic plants and kits, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as barren fields, hazards of higher-end species, damage of non-target organisms such as earthworms, etc.
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Embodiment 1
[0051] The design and synthesis of embodiment 1 primer probe
[0052] For the double-stranded RNA that triggers specific gene silencing in RNAi transgenic corn DBN11061, design dPCR forward and reverse primers and probes, as shown in Table 1, the nucleotide sequence of the DBN11061 double-stranded RNA forward primer is shown in SEQ ID NO:1 , the nucleic acid sequence of the reverse primer is shown in SEQ ID NO:2, the nucleic acid sequence of the probe is shown in SEQ ID NO:3, synthesized by Thermo Fisher Scientific (China) Co., Ltd.
[0053] Table 1 Primer and Probe Sequences
[0054]
Embodiment 2
[0055] Example 2 Method optimization based on RNAi transgenic corn total RNA extraction
[0056] During the extraction process, the leaves are first ground into powder using liquid nitrogen, and then different kits PureLink TM RNA Mini Kit (ET-Kit1, Thermo Fisher Scientific (China) Co., Ltd.), Column Plant Total RNA Extraction and Purification Kit (ET-Kit2, Sangon Bioengineering (Shanghai) Co., Ltd.) and MiniBEST Plant RNAExtraction Kit (ET-Kit3, Treasure Bioengineering (Dalian) Co., Ltd.) was used to extract total RNA from the leaves of RNAi-based transgenic maize, and the obtained RNA was stored at -80°C. cDNA, and follow QuantStudio TM The default program of the 3D digital PCR instrument was used to perform digital PCR and compare the results. The experiments were repeated 3 times.
[0057] The principles of different plant total RNA extraction kits are basically similar. The extraction process mainly includes: (1) adding liquid nitrogen to fully grind to completely bre...
Embodiment 3
[0060] Example 3 Method optimization for reverse transcription of RNA into cDNA
[0061] Transgenic maize leaf RNA obtained using ET Kit 3 was obtained using different kits, ProtoScript cDNA First Strand Synthesis Kit (RT Kit A, NEB (Beijing) Co., Ltd.), PrimeScript TM II1st Strand cDNA Synthesis Kit (RT Kit B, Treasure Bioengineering (Dalian) Co., Ltd.) and Maxima H Minus First Strand cDNA Synthesis Kit (RT Kit C, Thermo Fisher Scientific (China) Co., Ltd.) for reverse transcription, obtained cDNA according to QuantStudio TM The 3D default program was used to perform digital PCR and compare the results, and the experiments were repeated 3 times.
[0062] Reverse transcription refers to the process of using RNA as a template to synthesize the first strand of cDNA under the action of reverse transcriptase, specifically: (1) react at 65°C for different times, and cool on ice to denature the RNA and open its second strand. (2) add random primers, dNTPs, buffer (buffer) and re...
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