CN114317775A - Application of RNA m6A modification of NCOA4 as gamma-ray radiation marker - Google Patents
Application of RNA m6A modification of NCOA4 as gamma-ray radiation marker Download PDFInfo
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Abstract
The invention discloses application of a modification level or a relative modification level of RNA m6A of NCOA4 in preparation of products for detecting or assisting in detecting whether gamma-ray radiation is applied. The application of the RNA m6A modification of NCOA4 as the gamma-ray radiation marker can detect or assist to detect whether the object to be detected is irradiated by gamma rays more accurately, quickly and sensitively.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of RNA m6A modification of NCOA4 as a gamma-ray radiation marker.
Background
Accurate, long-term diagnosis of radiation victims in nuclear radiation accidents and nuclear disasters is an important basis for accident rescue and medical treatment. Currently, biological methods are available for detecting exposure to gamma radiation, including clinical indications, peripheral blood lymphocyte counts, and cytogenetic techniques. The expression quantity of the protein gamma-H2 AX is a radiation marker with high specificity, is a common biological dosimeter, and plays a role of 'gold standard' in the detection of various radiation samples.
However, since the double-strand break can be repaired quickly, the expression level of γ -H2AX gradually disappears with time delay, and thus cannot be used for dose estimation in the late stage of irradiation. Therefore, there is a need for a stable long-lasting biomarker.
Disclosure of Invention
The invention aims to provide application of RNA m6A modification of NCOA4 as a gamma-ray radiation marker, which can detect or assist in detecting whether a to-be-detected object is irradiated by gamma rays more accurately, quickly and sensitively.
To achieve the above object, the present invention provides the use of the modification level or relative modification level of RNA m6A of NCOA4 in the preparation of a product for detecting or aiding in the detection of whether it is exposed to gamma radiation.
Use of a system for detecting or aiding in the detection of whether a NCOA4 has been subjected to gamma radiation, of an RNA m6A modification as a marker, in the manufacture of a product for detecting or aiding in the detection of whether a NCOA4 has been subjected to gamma radiation.
A system for detecting the modification level or relative modification level of RNA m6A of NCOA4, wherein the system for detecting the modification level or relative modification level of RNA m6A of NCOA4 is a system for detecting the modification level or relative modification level of RNA m6A of NCOA4 by using an RNA m6A chip technology or a system for detecting the modification level or relative modification level of RNA m6A of NCOA4 by using a MeRIP-qPCR technology.
Preferably, the system for detecting the level of modification or relative modification of RNA m6A of NCOA4 comprises primers and/or reagents and/or kits and/or apparatus.
Preferably, the system for detecting the modification level or relative modification level of the RNA m6A by MeRIP-qPCR comprises primers, a kit and/or other reagents and/or instruments required for performing MeRIP-qPCR.
Preferably, the system for detecting the modification level or relative modification level of RNA m6A further comprises a data processing system for converting the modification level or relative modification level of RNA m6A from a test object into a detection result of the test object.
The data processing system comprises a data input module, a data comparison module and a conclusion output module; the data input module is used for inputting the numerical value of the modification level or the relative modification level of the RNA m6A of the NCOA4 of a to-be-detected object; the data comparison module is used for comparing the RNA m6A modification level or relative modification level of the NCOA4 of a to-be-detected object with an object which is not subjected to gamma ray radiation; the conclusion output module is used for outputting a conclusion: if the modification level or relative modification level of RNA m6A of NCOA4 of the object to be tested is greater than the modification level or relative modification level of RNA m6A of NCOA4 of the object not subjected to gamma-ray radiation, concluding that the object to be tested is subjected to gamma-ray radiation; if the level of modification or relative modification of RNA m6A of NCOA4 of the test object is less than or equal to the level of modification or relative modification of RNA m6A of NCOA4 of the test object that has not been subjected to gamma-ray irradiation, then it is concluded that the test object has not been subjected to gamma-ray irradiation.
Preferably, the RNA m6A modification level or relative modification level of the NCOA4 gene is the RNA m6A modification level or relative modification level of the NCOA4 gene in blood or cells.
Preferably, the modification level or relative modification level of RNA m6A of the NCOA4 gene is the modification level or relative modification level of RNA m6A of NCOA4/NCOA4 gene in peripheral blood mononuclear cells isolated from peripheral blood of a mouse or Hela cells of a human.
Preferably, the primers for amplifying mouse peripheral blood mononuclear cells Ncoa4 consist of 2 primer pairs, and include primer pair 1 consisting of two single-stranded DNAs shown in Seq ID No.1 and Seq ID No.2, and primer pair 2 consisting of two single-stranded DNAs shown in Seq ID No.3 and Seq ID No. 4.
Preferably, the primer for amplifying NCOA4 of Hela cells is composed of 2 primer pairs, and includes primer pair 1 composed of two single-stranded DNAs shown by Seq ID No.5 and Seq ID No.6, and primer pair 2 composed of two single-stranded DNAs shown by Seq ID No.7 and Seq ID No. 8.
Preferably, the RNA m6A modification level or relative modification level of Ncoa4/Ncoa4 is the percentage of m6A modification after enrichment with m6A antibody (m6A-IP) relative to the amount of RNA (Input) by RNA m6A modification of Ncoa4/Ncoa 4.
Preferably, said exposure to gamma radiation means exposure to cobalt 60-gamma radiation.
The present invention proposes the characteristic of the change in the level of modification or relative level of RNA m6A of Ncoa4 with gamma-ray irradiation dose and the characteristic of the change in the level of modification or relative level of modification of RNA m6A of Ncoa4 with time at the same irradiation dose, which are used to evaluate the dose of gamma-ray irradiation or the time after irradiation of a subject to be tested.
The level of modification or relative level of modification of RNA m6A of Ncoa4 tended to increase first and then decrease within 1, 3, 7, 14, 28 days, reaching a maximum at 14 days after irradiation, compared to the normal group not subjected to gamma-ray irradiation; when the irradiation dose was 0.5Gy or more, the modification level or relative modification level of RNA m6A of Ncoa4 was significantly increased within 7 days after irradiation, as compared with the normal group not subjected to gamma-ray irradiation; the level of modification or relative modification of RNA m6A of Ncoa4 was significantly increased within 1-28 days when the radiation dose was ≧ 2Gy, compared to the normal group which did not undergo gamma-ray radiation.
Therefore, the invention adopts the application of the RNA m6A modification of NCOA4 as a gamma-ray radiation marker, and the modification level or the relative modification level of the RNA m6A of NCOA4 can detect whether the object to be detected is subjected to gamma-ray radiation and the evaluation of the radiation dose, thereby being beneficial to timely carrying out accident rescue and wounded treatment and having important significance on radiation treatment.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
FIG. 1 is a graph showing the results of MeRIP-qPCR experiments on mouse peripheral blood mononuclear cells treated in different batches to detect the level of modification or relative modification of RNA m6A of Ncoa 4;
FIG. 2 is a graph showing the results of MeRIP-qPCR assay for different batches of treated Hela cells to detect the level of modification or relative modification of RNA m6A of NCOA 4.
Detailed Description
The technical solution of the present invention is further illustrated by the accompanying drawings and examples.
Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified, and examples of the examples are shown in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
The sources of materials, reagents and assay techniques used in the following examples are as follows:
the mouse RNA m6A-Seq chip was provided by Shanghai Kangcheng bioinformatics technology, Inc.
M6A antibody [ m6A (D9D9W) Rabbit mAb required for MeRIP experiments, cat #: 56593]IgG (Normal rabbit IgG, product number: 2729s) is a product of CST company in America; the required Protein A/G PLUS-Agarose is a product of Santa Cruz, USA, with the following code: sc-2003; fluorescent quantitative detection Kit (KAPA)
FAST qRT-PCR kit) is a product of KAPA Biosystems, usa under the following product numbers: KK 4601.
Example one
Level of RNA m6A modification of Ncoa4 Gene after irradiation
Male C57BL/6N mice 6-8 weeks old are from Beijing Wintolite laboratory animal technology, Inc., and C57BL/6N mice in good health state are irradiated with 6.5Gy of cobalt 60-gamma rays, then RNA is extracted after peripheral blood mononuclear cells obtained by peripheral blood separation are taken at different time points (0d, 1d, 3d, 7d, 14d and 28d), and RNA m6A chip experiments are carried out on 6 groups of total RNA samples. The RNA m6A chip experiment was performed using an m6A-mRNA & lncRNA chip.
The quality of the total RNA of the peripheral blood mononuclear cells is detected before the sequencing of the RNA m6A chip, the detected RNA quality is good, and the RNA is randomly broken into fragments of about 100 nt. Co-incubating the specific antibody modified by the RNA m6A and the RNA fragment, and grasping the methylation modified fragment for sequencing; at the same time, a control (Input) sample is sequenced in parallel, the control sample only contains the broken RNA fragment and does not add RNA methylation specific antibody to incubate with the RNA methylation specific antibody.
The m6A chip is scanned by a scanner, data signal extraction, normalization processing and m6A signal difference peak analysis are carried out, and the change of RNA m6A modification before and after irradiation is compared, and the results show that the RNA modified and up-regulated by m6A at different time points after irradiation is 110 on the first day, 1487 on the third day, 74 on the seventh day, 183 on the fourteenth day and 45 on the twenty-eight day respectively, the RNA modified and down-regulated by m6A is 1843 on the first day, 244 on the third day, 865 on the seventh day, 3819 on the fourteenth day and 317 on the twenty-eight day respectively, the RNA m6A modification of the Ncoa4 gene which is obviously up-regulated at 5 time points after irradiation (as shown in Table 1) is screened, namely the RNA m6A of the Ncoa4 is modified to be used as a marker molecule for gamma ray irradiation detection, the mRNA sequence number of the Ncoa 4: ENSMUST00000169722, can detect whether the object to be detected is exposed to gamma ray through the RNA m6A modification level or relative modification level of Ncoa4 gene.
TABLE 1 RNA m6A chip analysis results of Ncoa4 gene after mouse peripheral blood mononuclear cell irradiation
Example two
Verification of the level of modification of RNA m6A of Ncoa4 gene before and after irradiation with RNA m6A chip
Male C57BL/6N mice 6-8 weeks old are from Beijing Wintonli Hua laboratory animal technology Co., Ltd, and healthy C57BL/6N mice are irradiated before (control group) or irradiated with 2Gy cobalt 60-gamma ray, and then RNA is extracted after peripheral blood mononuclear cells obtained by peripheral blood separation are taken at different time points (1d, 14d), and 3 groups of total RNA samples are subjected to RNA m6A chip experiments. The RNA m6A chip experiment was performed using an m6A-mRNA & lncRNA chip.
The quality of the total RNA of the peripheral blood mononuclear cells is detected before the sequencing of the RNA m6A chip, the detected RNA quality is good, and the RNA is randomly broken into fragments of about 100 nt. Co-incubating the specific antibody modified by the RNA m6A and the RNA fragment, and grasping the methylation modified fragment for sequencing; at the same time, a control (Input) sample is sequenced in parallel, the control sample only contains the broken RNA fragment and does not add RNA methylation specific antibody to incubate with the RNA methylation specific antibody. Scanning the m6A chip by a scanner, extracting data signals, carrying out normalization processing and m6A signal difference peak analysis, comparing the modification change of the RNA m6A before and after irradiation, and displaying that the modified and up-regulated RNAs of m6A are 88 RNAs on the first day and 248 RNAs on the fourteenth day at different time points after irradiation; the modified downregulated RNA of m6A was 43 on the first day and 550 on the fourteenth day, respectively. RNA m6A modification of Ncoa4 gene that was more clearly upregulated at 2 time points after irradiation (as shown in table 2) was screened by comparison, indicating that mice subjected to irradiation with gamma rays had a significantly increased level of RNA m6A modification or relative level of modification of Ncoa4 gene in peripheral blood mononuclear cells relative to mice not subjected to irradiation with gamma rays.
TABLE 2 RNA m6A chip analysis results of Ncoa4 gene after mouse peripheral blood mononuclear cell irradiation
EXAMPLE III
Validation of the level of modification of RNA m6A of the Ncoa4 gene in peripheral blood mononuclear cells of mice using the MeRIP-qPCR experiment
1. Mice were irradiated with cobalt 60-gamma radiation and grouped
Male 6-8 week-old C57BL/6N mice were obtained from Beijing Wittingle laboratory animal technology, Inc., and healthy C57BL/6N mice were given cobalt 60-gamma irradiation. The irradiation conditions were room temperature, the irradiation distance was 3m, and the dose rate was 69.1 cGy/min. Mice were grouped according to the irradiation dose and the time to isolate peripheral blood mononuclear cells after irradiation, 10 mice per group, and the specific group is shown in table 3.
TABLE 3 irradiation dose and mouse grouping
2. Isolation of peripheral blood mononuclear cells
Blood was collected from mouse orbit and peripheral blood mononuclear cells were isolated using mouse peripheral blood lymphocyte isolate KIT (brand: TBD; cat # LTS 1092-KIT). The method comprises the following specific steps:
1) 0.5mL of anticoagulated blood (blood is easy to separate after being placed for a long time and needs to be premixed) is taken, and 0.5mL of diluent is added and mixed evenly. 3mL of the separation solution is added into a 15mL centrifuge tube, the centrifuge tube is inclined at an angle of 45 degrees, and the diluted peripheral blood is slowly added into the centrifuge tube containing the separation solution along the tube wall.
2) The tube was placed in a centrifuge and centrifuged at 500g for 20 min. The blood cells are divided into 4 layers in a centrifuge tube, the white lymphocyte layer (the upper layer is not absorbed, the lower layer can be absorbed) is carefully absorbed, the white lymphocyte layer is moved into a new centrifuge tube, cleaning solution or PBS is added to 10mL, and a dropper is used for uniformly mixing.
3) Centrifugation is carried out at 1100rpm for 15min, the supernatant is discarded, resuspension is carried out with 1mL of washing solution or PBS, 20. mu.L of the cell is taken for cell counting, the remaining cells are added into a new 1.5mL of EP tube, the supernatant is discarded after centrifugation is carried out at 1100rpm for 15min, and resuspension is carried out with 1mL of Trizol.
3. RNA extraction of peripheral blood mononuclear cells
The method for extracting RNA by using phenol chloroform comprises the following specific steps:
1) to Trizol resuspended peripheral blood mononuclear cells was added 200. mu.L of chloroform, shaken vigorously for 15s, and allowed to stand for 5min until liquid separation.
2) Centrifuge at 12000g for 15min at 4 ℃.
3) Sucking supernatant (avoiding sucking liquid on other layers) to a new EP tube as much as possible, adding isopropanol with the same volume, shaking, mixing uniformly, and incubating at-20 deg.C for 1 h.
4) Centrifuge at 14000rpm for 30min at 4 ℃ and discard the supernatant.
5) 1mL of 75% ethanol pre-cooled in advance was added, vortexed, centrifuged at 14000rpm for 3min at 4 ℃ and the supernatant was discarded.
6) And repeating the steps.
7) The EP tube was inverted onto absorbent paper, air dried at room temperature for 10min, and 50. mu.L of Nase free water was added to resuspend the pellet.
8) RNA was quantified by determining the total RNA concentration using a ultramicro spectrophotometer Nano-300 and assessing the purity by the ratio A260nm/A280 nm.
4. Fragmentation of RNA:
1) the concentration of the above RNA was adjusted to 10 ng/. mu.L with RNase free water and the total amount of RNA was ensured to be at least 2. mu.g.
2) Pipetting 200. mu.L of the RNA sample into a fresh EP tube, adding 2. mu.L of RNase inhibitor using Thermo Fisher Scientific LabServTMThe Model 120 ultrasonicator performs ultrasonication with power of 20%, ultrasonication for 1s, and interval of 2s for 15 times.
3) Sucking 20 mu L of the RNA sample after ultrasonic treatment into a new EP tube, marking the RNA sample as Input, and storing the RNA sample at-80 ℃ for later use;
4) the remaining post-sonication RNA samples were placed on ice for IP experiments.
5. MeRIP experiment:
1) preparing a reagent:
IP reaction solution: 50mmol/L Tris-HCl (pH 7.4), 150mmol/L NaCl, 0.1% NP-40;
1 × IP buffer: 10mM Tris-HCl (pH 7.4), 150mM NaCl, 0.1% NP-40;
1 × washing buffer: 10mM Tris-HCl (pH 7.4), 50mM NaCl, 0.1% NP-40;
elution buffer: 10mM Tris-HCl (pH 7.4), 1mM EDTA, 0.05% SDS.
(Note: the above solutions were all prepared with RNase free water)
2) Incubation of beads with antibody:
dividing the experiment into an m6A antibody IP group and an IgG IP group for comparison, marking a required 1.5mLEP tube, respectively adding 20 mu L of Protein A/G PLUS-Agarose, adding 500 mu L of IP reaction liquid, shaking, uniformly mixing, centrifuging at 4 ℃ and 5000rpm for 2min, cleaning beads, discarding supernatant, cleaning for 2 times, adding 500 mu L of IP reaction liquid, adding 1 mu G of m6A antibody into the m6A antibody IP group, adding 1 mu G of IgG antibody into the IgG IP group, rotating and incubating for 4h by a shaking table at 4 ℃, centrifuging at 4 ℃ and 5000rpm for 2min, cleaning beads, discarding supernatant, adding 500 mu L of IP reaction liquid, and repeating the cleaning steps to obtain antibody coupled beads.
3) RNA immunoprecipitation:
adding 500 mu L of IP reaction liquid into antibody-coupled beads, respectively adding 90 mu L of fragmented RNA into an m6A antibody IP group and an IgG IP group, carrying out rotary incubation at 4 ℃ overnight, centrifuging at 4 ℃ and 5000rpm for 2min, discarding the supernatant, adding 500 mu L of IP reaction liquid to clean the RNA which is not combined with the beads, shaking and uniformly mixing, centrifuging at 4 ℃ and 5000rpm for 2min, discarding the supernatant, totally cleaning for 3 times, then adding 500 mu L of washing buffer solution to clean the beads, shaking and uniformly mixing, centrifuging at 4 ℃ and 5000rpm for 2min, discarding the supernatant, and totally cleaning for 3 times.
4) Elution and purification of m6A modified RNA:
to the above sample was added 100. mu.L of elution buffer, 3. mu.L of proteinase K, and the mixture was incubated at 50 ℃ for 30min with rotation, and the eluted RNA was again purified by phenol chloroform method and finally dissolved in 20. mu.L of LRNase free water.
6. Reverse transcription of RNA from peripheral blood mononuclear cells to cDNA
The kit used for reverse transcription is a MonScriptTM RTIII AII-in-One Mix reverse transcription kit, and the specific steps are as follows:
1) and taking out the components from the kit, dissolving the components on ice, mixing the dissolved components uniformly, centrifuging the mixture for a short time, and placing the mixture on ice for later use.
2) Reverse transcription systems were prepared in 200. mu.L RNase-free inlet PCR tubes as shown in Table 4.
TABLE 4 reverse transcription System (20. mu.L)
3) After mixing uniformly, putting the mixture into a PCR instrument for reverse transcription reaction. The PCR program was set as: the first step is at 55 ℃ for 15 min; the second step, 85 ℃, 5 min; the third step is at 4 ℃ for 10 min.
4) The cDNA product after reverse transcription was removed and diluted with 100. mu.L of RNase free water.
7、qPCR
1) Specific primers were designed for the potential site of RNA m6A modification of mouse Ncoa4, with the primer sequences as shown in table 5:
TABLE 5 real-time fluorescent quantitative PCR primer sequences
2) The kit used for real-time quantitative PCR is KAPA
The FAST qRT-PCR kit samples according to the system shown in Table 6, and is subpackaged in a 96-well plate, and each sample is subjected to three repeated reaction systems, namely three groups of parallel tests.
TABLE 6 qPCR sample application System
3) Centrifuging a 96-well plate at 3000rpm for 5min, putting the plate into a qPCR instrument for PCR reaction, and setting a qPCR program as follows: the first step is at 95 ℃ for 5 min; the second step, 95 ℃ and 5 s; the third step is at 60 ℃ for 30 s; the second step to the third step total 40 cycles; the fourth step: a dissolution profile; the fifth step: storing at 4 ℃.
4) The data were analyzed and the m6A modification level or relative modification level of the RNA was calculated according to the following formula comparing the difference in% Input between groups.
Calculating the formula: Δ Ct ═ Ct (rip) - [ Ct (input) -log2(dilution factor)]Then m6A modification level% Input 2 of RNA-△Ct。
The m6A modification level or relative modification level of RNA of Ncoa4 three mean data after 3 MeRIP-qPCR experiments are shown in fig. 1, where a is the determination of the amount of RNA required for MeRIP-qPCR experiments to detect the m6A modification level or relative modification level of RNA of Ncoa4, and when the RNA required for m6A antibody IP is greater than or equal to 2 μ g, the RNA m6A modification level or relative modification level of Ncoa4 is compared between the irradiated group (6.5Gy irradiation dose, 1 day after irradiation) and the normal group (not subjected to gamma radiation), x p < 0.0001; primer 1(346-480) with B Ncoa4 detected a change in the level of modification or relative level of modification of RNA m6A at different irradiation doses and time points Ncoa4, at which the level of modification or relative level of modification of RNA m6A of Ncoa4 was compared with that of normal group (not subjected to gamma irradiation) for the irradiated group (0.2Gy, 0.5Gy, 1Gy, 2Gy, 4Gy, 6.5Gy, 10 Gy), respectively, { p < 0.01 } { p < 0.001 } p < 0.0001); primer 2(658 + 847) with C Ncoa4 detected a change in the level of modification or relative level of modification of RNA m6A at different doses of radiation and at time points Ncoa4, at which the level of modification or relative level of modification of RNA m6A of Ncoa4 was compared with that of normal group (not subjected to gamma radiation) for the irradiated groups (0.2Gy, 0.5Gy, 1Gy, 2Gy, 4Gy, 6.5Gy, 10 Gy), p < 0.1, p < 0.01, p < 0.001, p < 0.0001).
Both pairs of primers modified with RNA m6A for detection of Ncoa4 indicated: RNA m6A modification of Ncoa4 was significantly increased with increasing radiation dose, gradually increased over 1-14 days and gradually decreased over 14-28 days; when the radiation dose was not less than 0.5Gy, the RNA m6A modification level or relative m6A modification level of Ncoa4 gene was significantly increased within 7 days after irradiation; when the irradiation dose was 2Gy or more, the level of modification of RNA m6A or the level of modification of the gene Ncoa4 relative to m6A was significantly higher in 1 to 28 days than in the group not subjected to gamma-ray irradiation. The RNA m6A modification of the Ncoa4 gene can respond to ionizing radiation stimulation in a short time (1 day) and is suitable for detecting the irradiation dose of more than or equal to 0.5 Gy; meanwhile, when the irradiation dose is higher, the RNA m6A modification of the target gene can maintain a high level for a longer time (28 days), and is suitable for dose evaluation for a longer time after irradiation.
Example four
Validation of the level of modification of RNA m6A of the NCOA4 gene in human Hela cells Using the MeRIP-qPCR experiment
1. Cell culture
Hela cell is human cervical cancer cell, cultured in DMEM medium containing 10% fetal calf serum, and placed with 5% CO2Subculturing in an incubator at 37 ℃.
2. Irradiating with cobalt 60-gamma ray
Hela cells in good growth state were irradiated with cobalt 60-gamma rays. Wherein the irradiation condition of the cobalt 60-gamma ray irradiation is room temperature, the irradiation dose is 10Gy, the irradiation distance is 3 meters, and the dose rate is 69.1 cGy/min. Cells from the control (non-irradiated) group (labeled Ctrl) were harvested, followed by cells 1 hour after 10Gy irradiation (labeled IR).
3. RNA extraction of Hela cells
RNA was extracted by phenol chloroform method.
4. Fragmentation of RNA
5. MeRIP experiment
6. Reverse transcription of RNA from Hela cells into cDNA
The kit used for reverse transcription is a MonScriptTM RTIII AII-in-One Mix reverse transcription kit, and the reverse transcription system is shown in Table 4.
7、qPCR：
1) Specific primers were designed for the potential site of RNA m6A modification of the human NCOA4 gene, and the primer sequences are shown in table 7.
TABLE 7 real-time fluorescent quantitative PCR primer sequences
2) The kit used for real-time quantitative PCR is KAPA
The FAST qRT-PCR kit samples according to the system shown in Table 6, and is subpackaged in a 96-well plate, and each sample is subjected to three repeated reaction systems, namely three groups of parallel tests.
3) Centrifuging a 96-well plate at 3000rpm for 5min, putting the plate into a qPCR instrument for PCR reaction, and setting a qPCR program as follows: the first step is at 95 ℃ for 5 min; the second step, 95 ℃ and 5 s; the third step is at 60 ℃ for 30 s; the second step to the third step total 40 cycles; the fourth step: a dissolution profile; the fifth step: storing at 4 ℃.
4) The data were analyzed and the m6A modification level or relative modification level of the RNA was calculated according to the following formula comparing the difference in% Input between groups.
Calculating the formula: Δ Ct ═ Ct (rip) - [ Ct (input) -log2(dilution factor)]Then m6A modification level% Input 2 of RNA-△Ct。
The m6A modification level or relative modification level of NCOA4 RNA is shown in fig. 2 as an average of three data after 3 MeRIP-qPCR experiments, where primer 1(219 × 319) with a being NCOA4 detects the m6A modification level or relative modification level of NCOA4, the RNA m6A modification level or relative modification level of NCOA4 is compared between the irradiated group (10Gy irradiation dose, 1 hour after irradiation) and the normal group (not subjected to gamma irradiation) × p < 0.001; primer 2(796-897) with B as NCOA4 detects the modification level or relative modification level of RNA m6A of NCOA4, and the modification level or relative modification level of RNA m6A of NCOA4 is compared with that of normal group (not subjected to gamma-ray radiation) in the irradiation group (10Gy irradiation dose, 1 hour after irradiation).
Compared with the non-irradiation (0Gy), the irradiation dose of 10Gy has a significant difference in the m6A modification level or relative modification level of the RNA of NCOA4, which indicates that whether the object to be tested is irradiated by gamma rays can be detected by detecting the m6A modification level or relative modification level of the RNA of NCOA 4.
The invention analyzes genes with obvious difference of RNA m6A-seq after RNA m6A modification after irradiation through a plurality of analysis software and algorithms, finds that the RNA m6A modification level or relative modification level of Ncoa4 is obviously increased at a plurality of time points after irradiation, further utilizes MeRIP-qPCR in an independent mouse peripheral blood mononuclear cell sample queue, finds that the RNA m6A modification of Ncoa4 after gamma-ray irradiation treatment changes along with the change rule of irradiation dose and time after irradiation, further utilizes MeRIP-qPCR in human Hela cells, and finds that the RNA m6A modification level or relative modification level of NCOA4 after gamma-ray irradiation treatment is obviously increased compared with a group not subjected to gamma-ray irradiation.
Furthermore, the change characteristics of the RNA m6A modification after gamma-ray irradiation treatment were clarified, and it was found that the RNA m6A modification level of Ncoa4 was significantly increased at various time points after gamma-ray irradiation treatment, and the RNA m6A modification level of the gene was used for detecting gamma-ray irradiation.
Therefore, the invention adopts the application of the RNA m6A modification of NCOA4 as a gamma-ray radiation marker, and the modification level or the relative modification level of the RNA m6A of NCOA4 can detect whether the object to be detected is subjected to gamma-ray radiation and the evaluation of the radiation dose, thereby being beneficial to timely carrying out accident rescue and wounded treatment and having important significance on radiation treatment.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.
Claims (10)
1. Use of a level of modification or relative modification of RNA m6A of NCOA4 in the preparation of a product for detecting or aiding in the detection of exposure to gamma radiation.
2. Use of a system for detecting or aiding in the detection of whether a NCOA4 has been subjected to gamma radiation, of an RNA m6A modification as a marker, in the manufacture of a product for detecting or aiding in the detection of whether a NCOA4 has been subjected to gamma radiation.
3. A system for detecting the level of modification or relative level of modification of RNA m6A of NCOA4, comprising: the system for detecting the modification level or relative modification level of RNA m6A of NCOA4 is a system for detecting the modification level or relative modification level of RNA m6A of NCOA4 by using an RNA m6A chip technology or a system for detecting the modification level or relative modification level of RNA m6A of NCOA4 by using a MeRIP-qPCR technology.
4. The system for detecting the modification level or relative modification level of RNA m6A of NCOA4 according to claim 3, wherein: the system for detecting the modification level or relative modification level of the RNA m6A of the NCOA4 comprises primers and/or reagents and/or kits and/or instruments.
5. The system for detecting the modification level or relative modification level of RNA m6A of NCOA4 according to claim 3, wherein: the system for detecting the modification level or relative modification level of the RNA m6A by using MeRIP-qPCR comprises primers, a kit and/or other reagents and/or instruments required for carrying out MeRIP-qPCR.
6. The system for detecting the modification level or relative modification level of RNA m6A of NCOA4 according to claim 3, wherein: the system for detecting the level of modification or relative level of modification of RNA m6A further comprises a data processing system for converting the level of modification or relative level of modification of RNA m6A from a test subject into a detection result of the test subject.
7. The system for detecting the modification level or relative modification level of RNA m6A of NCOA4 according to claim 3, wherein: the modification level or relative modification level of RNA m6A of the NCOA4 gene is the modification level or relative modification level of RNA m6A of the NCOA4 gene in blood or cells.
8. The system for detecting the modification level or relative modification level of RNA m6A of NCOA4 according to claim 7, wherein: the modification level or relative modification level of RNA m6A of the NCOA4 gene is the modification level or relative modification level of RNA m6A of the Ncoa4/NCOA4 gene in peripheral blood mononuclear cells isolated from peripheral blood of a mouse or Hela cells of a human.
9. The system for detecting the modification level or relative modification level of RNA m6A of NCOA4 according to claim 8, wherein: the primers for amplifying mouse peripheral blood mononuclear cells Ncoa4 consisted of 2 primer pairs, and included primer pair 1 consisting of two single-stranded DNAs shown in Seq ID No.1 and Seq ID No.2, and primer pair 2 consisting of two single-stranded DNAs shown in Seq ID No.3 and Seq ID No. 4.
10. The system for detecting the modification level or relative modification level of RNA m6A of NCOA4 according to claim 8, wherein: the primers for amplifying NCOA4 of Hela cells were composed of 2 primer pairs, including primer pair 1 composed of two single-stranded DNAs shown in Seq ID No.5 and Seq ID No.6, and primer pair 2 composed of two single-stranded DNAs shown in Seq ID No.7 and Seq ID No. 8.
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