m6a RNA methylation site detection method
A detection method and methylation technology, applied in the field of molecular biology, can solve problems such as restricting research and hindering the understanding of human diseases, achieving the effects of high RNA integrity, reduced errors, and simple experimental process
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Embodiment 1
[0071] Example 1 Extraction of Trace Cell Total RNA
[0072] The present invention provides a method for detecting m6A methylation in a trace amount of total cellular RNA (see figure 1 ), providing a method for detecting m6A methylation sites in mRNA and IncRNA and its application.
[0073] The present invention adopts the provided ultra-trace rRNA strand-specific m6A methylation detection library construction kit, and the method includes but is not limited to one or more of the following steps:
[0074] 1. Sample pretreatment: the sample used in this example is 293-T cells. Firstly, the cultivated 10 6 Collect 293-T cells, centrifuge at 500×g for 5-8min at room temperature, discard the supernatant and leave the cell pellet.
[0075] 2. Genome-free total RNA extraction: Extract total RNA from 293-T cell samples using the column extraction method (QIAGEN, Cat. No. 217204). During the extraction process, pay attention to using it in advance to eliminate the genome in the tota...
Embodiment 2
[0084] Example 2 Capture of m6A RNA fragments
[0085] 1. Prepare antibody magnetic bead RNA mixture: take 10µg of total RNA as an example, prepare according to the following system, mix well by inverting up and down, and incubate at room temperature for 1.5 hours on a rotator;
[0086]
[0087] RNA sample concentration is 1µg / µL, m6A-specific antibody 2µg / tube;
[0088] 2. Lysis of total RNA: After the incubation, add 10 µL RNase inhibitor and 2 µL nuclease lyase to the mixture in the previous step, and lyse at room temperature for 2-4 minutes;
[0089] 3. Enrichment of m6A methylated RNA: after lysis, place it on a small magnetic stand for 2-3 minutes, then remove the supernatant by magnetic separation;
[0090] 4. Elution of non-m6A RNA fragments: Add 150 µL of low-salt buffer and high-salt buffer to wash 3-4 times;
[0091] 5. Purification of m6A RNA fragments:
[0092] 1) Prepare 20µL proteinase K solution / tube for the purified complex in low-salt buffer and high-sa...
Embodiment 3
[0096] Embodiment 3 m6A RNA library construction
[0097]The RNA products enriched in Example 2 were subjected to rRNA strand-specific library construction.
[0098] 1. Configure one-strand cDNA synthesis: configure on ice according to the following system;
[0099]
[0100] 1) Put the above mixture on ice immediately after running the following reaction procedure: 72°C, 3 minutes, 2 minutes on ice;
[0101] 2) After preparing the mixed solution according to the following system, take the Mix and add it to the reaction solution in the previous step:
[0102]
[0103] 3) After mixing by pipetting, run the following program: 90min at 42°C; 10min at 70°C; ∞ at 4°C.
[0104] 2. Prepare PCR1 reaction
[0105] 1) Configure the reaction mix according to the following reagents, mix by pipetting and add to the reaction solution in step 1:
[0106]
[0107] 2) Run the following program after brief centrifugation by pipetting and mixing: 94°C for 1min; 98°C for 15sec, 55°C f...
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