Transcriptome sequencing analysis method for differentially expressed genes of kidney injury after uranium contamination

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a transcriptome sequencing analysis method for differentially expressed genes of kidney injury after uranium contamination. Comprising the following steps: (1) grouping rats and establishing a kidney injury model after uranium contamination of the rats; (2) performing liquid nitrogen grinding and crushing on each group of kidney samples, and extracting and purifying to extract RNA (Ribonucleic Acid); (3) digesting the extracted qualified RNA, enriching mRNA, carrying out room temperature reaction to fragment the mRNA, and carrying out cDNA synthesis and connection; and (4) carrying out PCR reaction amplification on the connection product, cyclizing, and sequencing on a machine. And (5) performing principal component analysis on a sequencing result, and screening differential expression genes. By screening and identifying related genes after uranium acute exposure, a basis is provided for clinical treatment and marker screening of uranium-induced kidney injury, and a thought is provided for subsequent research on an action mechanism of uranium-induced kidney injury.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a transcriptome sequencing analysis method for differentially expressed genes in kidney damage after uranium poisoning. Background Art

[0002] With the widespread use of uranium, the population exposed to uranium is gradually expanding. Uranium has been proven to cause a certain degree of damage to renal function. The renal dysfunction caused by uranium is mainly related to renal proximal tubular epithelial cells, and in severe cases can lead to renal failure. A large number of studies have shown that the molecular mechanism of uranium-induced renal cell toxicity may involve mitochondrial dysfunction, oxidative stress, endoplasmic reticulum stress, etc.

[0003] Transcriptome sequencing technology (RNA-Seq) uses a new generation of high-throughput sequencing technology to study the expression pattern of eukaryotic cell genes by determining the sequence of transcription products and performing comparative analysis, providing accurate digital expression profile detection. RNA-Seq has become an important means of studying transcriptomes and gene expression analysis in the current biological field, with the characteristics of high detection throughput, strong sensitivity and good reproducibility.

[0004] At present, there are few studies on transcriptome sequencing analysis of differentially expressed genes in kidney damage after uranium exposure. The present invention intends to screen differentially expressed genes by analyzing the transcription products after acute uranium exposure, providing a basis for clinical treatment and marker screening of uranium-induced kidney damage. Summary of the invention

[0005] Technical issues

[0006] The purpose of the present invention is to provide a transcriptome sequencing analysis method for differentially expressed genes in kidney damage after uranium exposure, so as to be able to perform transcriptome sequencing analysis of kidney damage in rats after uranium exposure with high throughput, high sensitivity and rapidity.

[0007] Technical Solution

[0008] A transcriptome sequencing analysis method for differentially expressed genes in kidney damage after uranium exposure, characterized by comprising the following steps: (1) rats are grouped to establish a kidney damage model in rats after uranium exposure; (2) the kidney samples of each group are ground and crushed by liquid nitrogen, extracted and purified to extract RNA; (3) the extracted RNA is digested to enrich mRNA, the mRNA is fragmented by room temperature reaction, and then cDNA is synthesized and connected; (4) the connection products are amplified by PCR reaction, circularized, and then sequenced on a machine. (5) the sequencing results are subjected to principal component analysis to screen differentially expressed genes.

[0009] The transcriptome sequencing analysis method is characterized in that step (1) rats are grouped to establish a uranium-exposed rat renal injury model; the specific method is as follows:

[0010] 1) Male SD rats aged 12 to 13 weeks, weighing 440 to 520 g, were selected and adaptively raised for 1 week before the experiment, with 6 rats per cage. Food and water were freely available during the experiment.

[0011] 2) The animals were divided into a control group and two uranium exposure groups: a 3 mg / kg uranyl nitrate group and a 20 mg / kg uranyl nitrate group. The animals were exposed using a NE-C25S compressed nebulizer inhaler.

[0012] 3) On the 5th day after exposure, kidneys of 3 rats were randomly selected from each group and used for transcriptome sequencing.

[0013] Step (4) amplifies the ligation product by PCR reaction, circularizes it and then performs sequencing on a machine. The specific method is as follows:

[0014] 1) Perform PCR reaction on the ligation product: digest the U-labeled second-strand template with UDG enzyme and then perform PCR amplification to obtain a DNA library;

[0015] 2) Library testing: Select the appropriate testing method to conduct quality inspection on the DNA library according to product requirements.

[0016] 3) PCR product cyclization: After the PCR product is denatured into a single strand, it is cyclized to obtain a single-stranded circular DNA library. After digesting the uncyclized linear DNA molecules, the final library is obtained.

[0017] 4) On-machine sequencing: Single-stranded circular DNA molecules are replicated through rolling circles to form a DNA nanoball DNB containing multiple copies. The obtained DNBs are added to the mesh holes on the chip using high-density DNA nanochip technology and sequenced using the combined probe anchor polymerization technology cPAS.

[0018] Step (5) performs principal component analysis on the sequencing results, and the method for screening differentially expressed genes is: using DESeq2 software to screen differentially expressed genes with |log2FC|≥2 and Q-value<0.05 as screening conditions.

[0019] Beneficial Effects

[0020] The present invention provides a transcriptome sequencing analysis method for differentially expressed genes in uranium-exposed rats with posterior kidney damage, which can perform transcriptome sequencing analysis of uranium-exposed rats with posterior kidney damage in a high-throughput, high-sensitive and rapid manner, and screen out the common differentially expressed genes in uranium-exposed rats with posterior kidney damage.

[0021] Principal component analysis (PCA) was used to screen and identify genes related to kidney damage after acute uranium exposure. The results showed that the transcription products of the exposure group and the control group were significantly different, but the transcription products between the two exposure groups were relatively similar; 692 differentially expressed genes were screened out compared with the 3 mg / kg group and the control group, and 839 differentially expressed genes were screened out compared with the 20 mg / kg group and the control group, of which 538 were differentially expressed genes in total.

[0022] The present invention can further be used for screening of co-differentially expressed genes in kidney damage in rats exposed to uranium, and protein interaction network (PPI) analysis can be used to screen key differentially expressed genes, providing a basis for clinical treatment and marker screening of uranium-induced kidney damage, and providing ideas for subsequent research on the mechanism of action of uranium-induced kidney damage. DETAILED DESCRIPTION

[0023] The present invention provides a transcriptome sequencing analysis method for differentially expressed genes in kidney damage after uranium exposure, so as to be able to perform high-throughput, high-sensitivity and rapid transcriptome sequencing analysis of kidney damage in rats after uranium exposure. Specifically, it includes: (1) grouping rats to establish a kidney damage model for rats after uranium exposure; (2) grinding and crushing the kidney samples of each group with liquid nitrogen, extracting and purifying the extracted RNA; (3) digesting the extracted RNA to enrich the mRNA, fragmenting the mRNA at room temperature, and then synthesizing and connecting cDNA; (4) amplifying the connection products by PCR reaction, circularizing them, and then sequencing them on a machine. (5) Principal component analysis is performed on the sequencing results to screen differentially expressed genes. The specific implementation steps are as follows:

[0024] (I) Experimental steps

[0025] 1. The rats were divided into groups to establish the renal injury model after uranium exposure:

[0026] (1) Male SD rats aged 12 to 13 weeks, weighing 440 to 520 g, were selected and adaptively raised for 1 week before the experiment. Six rats were placed in each cage. Food and water were freely available during the experiment.

[0027] (2) The animals were divided into a control group and two uranium exposure groups: a 3 mg / kg uranyl nitrate group and a 20 mg / kg uranyl nitrate group, denoted as CON, U3, and U20. The animals were exposed using a NE-C25S compressed nebulizer inhaler.

[0028] (3) On the fifth day after exposure, kidneys of three rats were randomly selected from each group and used for transcriptome sequencing.

[0029] 2. Grind and crush the kidney samples in each group with liquid nitrogen, extract and purify RNA:

[0030] (1) Tissue sample fragmentation and lysis

[0031] Liquid nitrogen grinding and crushing: Take an appropriate amount of kidney tissue sample and grind it into powder under the protection of liquid nitrogen. Transfer it into an EP tube containing 1.5mL Trizol lysis solution and let it stand for 5 minutes to fully lyse the tissue cells.

[0032] (2) Sample extraction and purification

[0033] ① Centrifuge the ground and crushed tissue sample at 4°C and 12000g for 5 min, transfer the supernatant into an EP tube with 300 μL chloroform / isoamyl alcohol (24:1), invert and shake vigorously to mix, and then centrifuge at 4°C and 12000g for 8 min. If the middle layer is thick and the water phase is turbid, extract it again with an equal volume of chloroform / isoamyl alcohol (24:1) as the supernatant;

[0034] ② Pipette the supernatant into a new 1.5 mL centrifuge tube, add 2 / 3 of the supernatant volume of isopropanol, gently invert to mix, and place in a -20°C refrigerator for more than 2 hours;

[0035] ③ After standing, centrifuge at 4°C and 17500g for 25 min, discard the supernatant, wash with 0.9 mL 75% ethanol, invert the precipitate to suspend it, and centrifuge at 4°C and 17500g for 3 min (depending on the condition of the precipitate, wash it again with 75% ethanol and centrifuge at 4°C and 17500g for 3 min);

[0036] ④Discard the supernatant, centrifuge briefly and remove the remaining liquid, let it dry for 3-5 minutes, and dissolve the precipitate with 20-200μL DEPC water or RNase-free water.

[0037] (3) The concentration, purity (28S / 18S) and integrity (RIN value) of the total RNA were tested using a fully automatic capillary electrophoresis instrument to obtain qualified total RNA samples.

[0038] 3. Digest the extracted RNA to enrich the mRNA, react at room temperature to fragment the mRNA, and then synthesize and connect cDNA;

[0039] (1) mRNA isolation: A certain amount of qualified total RNA samples were digested with DNase I, and the digested total RNA samples were enriched for mRNA using Oligo(dT) magnetic beads.

[0040] (2) mRNA fragmentation: Add a fragmentation reagent to the enriched mRNA and react at room temperature to fragment the mRNA.

[0041] (3) cDNA synthesis: Prepare a first-strand synthesis reaction system to synthesize a first-strand cDNA. Add random primers to the fragmented mRNA to synthesize a first-strand cDNA: Prepare a second-strand synthesis reaction system to synthesize a second-strand cDNA. Use dUTP instead of dTTP, then perform end repair and add "A" to the amplified cDNA and connect it to the adapter.

[0042] 4. Perform PCR amplification and circularization on the ligation products and then sequence them on the machine:

[0043] (1) PCR reaction

[0044] The ligation product was subjected to PCR reaction, and the second-strand template labeled with U was digested with UDG enzyme and then PCR amplified to obtain a DNA library.

[0045] (2) Library detection

[0046] Perform quality control on the DNA library.

[0047] (3) PCR product cyclization

[0048] After the PCR product is denatured into a single strand, it is cyclized to obtain a single-stranded circular DNA library. After digesting the uncyclized linear DNA molecules, the final library is obtained.

[0049] (4) Sequencing

[0050] The single-stranded circular DNA molecule replicates through rolling circles to form a DNA nanoball (DNB) containing multiple copies. The resulting DNBs are added to the mesh holes on the chip using high-density DNA nanochip technology and sequenced using combined probe anchor polymerization technology (cPAS).

[0051] 5. Perform principal component analysis on sequencing results to screen differentially expressed genes

[0052] DESeq2 software was used to screen differentially expressed genes in the tested sequences with |log2FC|≥2 and Q-value<0.05 as the screening conditions to obtain the screened co-differentially expressed genes.

[0053] (II) Results Analysis

[0054] 1) The quality assessment of kidney high-throughput sequencing data and the comparison results with the reference genome are shown in Table 1. According to the experimental results, the unique comparison rate of all samples on the reference genome is higher than 90%. Therefore, the transcriptome sequencing results are reliable and of high quality and can be used for subsequent experimental analysis.

[0055] Table 1 Sequencing data quality processing and reference genome alignment results

[0056]

[0057] Note: (1) Rawreads: the number of raw sequencing data entries; (2) Cleanreads: the number of sequencing data entries after filtering; (3) M: reads unit, million; (4) Q20, Q30: the sequencing data after quality control evaluation, which respectively indicate the percentage of bases with base recognition accuracy above 99% and 99.9% in the total bases. Generally, the sequencing quality is considered good when Q20 ≥ 85% and Q30 ≥ 80%; (5) Alignment rate: the proportion of cleanreads with unique alignment positions on the reference genome.

[0058] 2) The results of principal component analysis (PCA) of gene expression showed that the three biological replicates of CON and U3 groups were highly similar, while the three biological replicates of U20 group were relatively less similar. In each treatment, CON and U3, U20 groups were clearly separated, while the separation between U3 and U20 groups was not obvious and there was crossover, indicating that the transcription products after 3mg / kg and 20mg / kg uranium exposure were similar, but the transcription products were significantly different from those in the control group.

[0059] 3) Differential genes were screened between CON and U3 groups, CON and U20 groups, and U3 and U20 groups based on the criteria of |log2FC|≥2 and Q-value<0.05. The differential genes between different samples were analyzed using DESeq2 software, and the number of differential genes in each group was counted. Compared with the control group, a total of 692 differential genes were screened in the U3 group, including 515 up-regulated genes and 177 down-regulated genes; compared with the control group, a total of 839 differential genes were screened in the U20 group, including 529 up-regulated genes and 310 down-regulated genes.

Claims

1. A transcriptome sequencing analysis method for differentially expressed genes in kidney damage after uranium exposure, characterized in that: The following steps are involved: (1) The rats were divided into groups to establish a model of renal injury after uranium exposure in rats; (2) The kidney samples of each group were ground and crushed with liquid nitrogen, and the RNA was extracted and purified; (3) The qualified RNA was digested and enriched for mRNA, and the mRNA was fragmented by room temperature reaction and then synthesized and connected for cDNA; (4) The ligation products were amplified by PCR reaction, circularized and sequenced on a machine; (5) The sequencing results were subjected to principal component analysis to screen differentially expressed genes.

2. The transcriptome sequencing analysis method according to claim 1, characterized in that: Step (1) Rats were divided into groups to establish a rat model of renal injury after uranium exposure; the specific method is as follows: 1) 12-13 week old male SD rats, weighing 440-520 g, were selected and adaptively raised for 1 week before the start of the experiment, with 6 rats in each cage, and food and water were freely available during the experiment; 2) The animals were divided into a control group and two uranium exposure groups: a 3 mg / kg group and a 20 mg / kg group with uranyl nitrate, and the animals were exposed using a NE-C25S compressed nebulizer; 3) On the fifth day after exposure, kidneys of three rats were randomly selected from each group and used for transcriptome sequencing.

3. The transcriptome sequencing analysis method according to claim 1 or 2, characterized in that: Step (4) amplifies the ligation product by PCR reaction, circularizes it and then performs sequencing on a machine. The specific method is as follows: 1) Perform PCR reaction on the ligation product: digest the U-labeled second-strand template with UDG enzyme and then perform PCR amplification to obtain a DNA library; 2) Library testing: Select the appropriate testing method to conduct quality inspection on the DNA library according to product requirements. 3) PCR product cyclization: After the PCR product is denatured into a single strand, it is cyclized to obtain a single-stranded circular DNA library. After digesting the uncyclized linear DNA molecules, the final library is obtained. 4) On-machine sequencing: Single-stranded circular DNA molecules are replicated through rolling circles to form a DNA nanoball DNB containing multiple copies. The obtained DNBs are added to the mesh holes on the chip using high-density DNA nanochip technology and sequenced using the combined probe anchor polymerization technology cPAS.

4. The transcriptome sequencing analysis method according to claim 1 or 2, characterized in that: Step (5) performs principal component analysis on the sequencing results, and the method for screening differentially expressed genes is: using DESeq2 software to screen differentially expressed genes with |log2FC|≥2 and Q-value<0.05 as screening conditions.

5. The transcriptome sequencing analysis method according to claim 3, characterized in that: Step (5) performs principal component analysis on the sequencing results, and the method for screening differentially expressed genes is: using DESeq2 software to screen differentially expressed genes with |log2FC|≥2 and Q-value<0.05 as screening conditions.

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