Application of SND1 and NDUFB2 as Internal Reference Genes in Quantitative Real-time PCR of Hepatocytes of Rana chensinensis

Abstract

The application discloses application of SND1 and NDUFB2 double-internal reference genes in fluorescence quantitative PCR of liver tissue of Leptophryne hamiltonii, in particular application of SND1 and NDUFB2 as double-internal reference genes in detection of expression of target genes in liver tissue of Leptophryne hamiltonii under exposure of sodium arsenite by fluorescence quantitative PCR, the nucleotide sequence of the SND1 is shown as SEQ ID NO. 1, the nucleotide sequence of the NDUFB2 is shown as SEQ ID NO. 2, and the target gene of the liver tissue of Leptophryne hamiltonii is a superoxide dismutase 1 gene. The SND1 gene and the NDUFB2 gene are stable in expression in liver samples of Leptophryne hamiltonii under exposure of sodium arsenite, and are suitable for being combined as internal reference genes for target gene expression correction.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically involving the application of SND1 and NDUFB2 dual internal reference genes in real-time quantitative PCR of liver tissue from the swamp frog. Background Technology

[0002] The marsh frog (Hylarana guentheri) is an amphibian closely related to aquatic environments. Amphibians possess both aquatic and terrestrial life histories, have highly permeable skin, and are sensitive to changes in pollutants in the aquatic environment, making them suitable for evaluating the toxic effects of pollutants on aquatic and wetland ecosystems. The liver is a vital organ in animals involved in metabolism, the transformation of exogenous compounds, detoxification, and oxidative stress defense; therefore, the liver of the marsh frog can serve as an important tissue for studying the molecular toxic effects of pollutants in the aquatic environment.

[0003] Arsenic is a highly toxic environmental pollutant with a certain degree of solubility and migration in aquatic environments. It can enter aquatic organisms through environmental media and adversely affect their metabolism, immunity, redox homeostasis, and growth and development. Sodium arsenite is a commonly used arsenic-containing compound in inorganic arsenic exposure toxicology studies. Detecting changes in the expression of liver-related genes in the marsh frog under different sodium arsenite exposure concentrations and exposure times is of great significance for evaluating the molecular toxic effects of arsenic pollution on amphibians and their response mechanisms. Real-time quantitative PCR (RT-qPCR) is a commonly used gene expression quantification technique with advantages such as high sensitivity, high specificity, wide detection range, and good reproducibility, and has been widely applied in environmental toxicology, animal physiology, and molecular biology research. When using RT-qPCR to analyze the relative expression levels of target genes, the detection results are easily affected by factors such as RNA quality, reverse transcription efficiency, primer specificity, amplification efficiency, and differences in sample processing. Therefore, it is necessary to select a stable internal reference gene to standardize and correct the target gene expression data to reduce systematic errors and improve the accuracy and comparability of the detection results.

[0004] Housekeeping genes such as ACTB, GAPDH, and TUBA, which are involved in basic cellular life activities, are often used as internal reference genes. However, the expression stability of internal reference genes is not constant and can be affected by factors such as species, tissue type, physiological state, type of pollutant, exposure concentration, and exposure time. Directly using unverified traditional internal reference genes may lead to deviations in the calculation of the relative expression level of the target gene, thereby affecting the accuracy and reliability of the detection results.

[0005] Currently, there is a lack of stable internal reference genes that have been systematically screened and validated for RT-qPCR analysis of the liver of the marsh frog under different sodium arsenite exposure concentrations and exposure times. Existing studies usually select traditional internal reference genes by referring to other species, other tissues, or other treatment conditions, which makes it difficult to guarantee their applicability in the sodium arsenite exposure system of marsh frog liver, thus limiting the accuracy, comparability, and reproducibility of target gene expression analysis results.

[0006] With the development of high-throughput transcriptome sequencing technology, candidate reference genes with relatively stable expression can be screened from the transcriptome based on gene expression abundance, intra-group variation, and inter-treatment differences in different samples. These genes are then validated using RT-qPCR and various stability evaluation methods. Compared to selecting reference genes solely from traditional housekeeping genes, this method broadens the candidate pool and improves the matching of the screened reference genes with specific species, tissues, and exposure conditions.

[0007] Therefore, it is necessary to screen and validate stable internal reference genes suitable for quantitative analysis of liver genes in the marsh frog under different sodium arsenite exposure concentrations and exposure times. Summary of the Invention

[0008] To address the lack of stable internal reference genes in existing technologies suitable for real-time quantitative PCR analysis of the liver tissue of the water frog under sodium arsenite exposure conditions, this invention provides the application of the SND1 and NDUFB2 dual internal reference genes in quantitative PCR of water frog liver tissue, especially the application of SND1 and NDUFB2 as dual internal reference genes in the detection of target gene expression in water frog liver tissue using quantitative PCR under sodium arsenite exposure conditions.

[0009] This invention screened and obtained the SND1 and NDUFB2 genes, and combined them as internal reference genes for RT-qPCR. This was used for the standardized correction of the expression levels of target genes in the liver of the marsh frog under sodium arsenite exposure conditions, so as to improve the accuracy, stability and reliability of the detection results.

[0010] The above-mentioned technical problem of the present invention can be achieved by the following technical solution: the application of SND1 and NDUFB2 dual internal reference genes in the real-time PCR of liver tissue of the water frog, especially the application of SND1 and NDUFB2 as dual internal reference genes in the real-time PCR detection of target gene expression in liver tissue of the water frog under sodium arsenite exposure conditions. The nucleotide sequence of SND1 is shown in SEQ ID NO.1, the nucleotide sequence of NDUFB2 is shown in SEQ ID NO.2, and the target gene of the liver tissue of the water frog is the superoxide dismutase 1 gene.

[0011] In the real-time fluorescence quantitative PCR analysis of some embodiments of the present invention, the geometric mean of the expression levels of the SND1 gene and the NDUFB2 gene is used as a standardization factor to correct the expression levels of target genes in the liver of the swamp frog.

[0012] Preferably, the primers for amplifying SND1 include the forward primer shown in SEQ ID NO.8 and the reverse primer shown in SEQ ID NO.9; the primers for amplifying NDUFB2 include the forward primer shown in SEQ ID NO.10 and the reverse primer shown in SEQ ID NO.11.

[0013] In some preferred embodiments of the present invention, the application of the SND1 and NDUFB2 dual internal reference genes in detecting the expression of target genes in the liver tissue of the swamp frog under sodium arsenite exposure conditions using real-time PCR includes the following steps: extracting total RNA from the liver sample of the swamp frog under sodium arsenite exposure conditions and reverse transcribing it to obtain cDNA; using the cDNA as a template, detecting the expression levels of the SND1 and NDUFB2 genes respectively using primers for amplifying SND1 and NDUFB2; and correcting the expression levels of the target genes in the liver of the swamp frog using the geometric mean of the expression levels of the SND1 and NDUFB2 genes as a standardization factor.

[0014] Preferably, the target gene in the liver of the swamp frog is a gene related to sodium arsenite exposure response, including but not limited to genes related to oxidative stress response.

[0015] In some embodiments of the present invention, the target gene in the liver of the swamp frog is the superoxide dismutase 1 gene (SOD1 gene).

[0016] Preferably, the relative expression level of the target gene in the liver of the swamp frog is determined using a 2-1 ratio. -ΔΔCt Calculation by method.

[0017] Preferably, the sodium arsenite exposure conditions include sodium arsenite exposure concentration and / or sodium arsenite exposure time.

[0018] Preferably, the sodium arsenite exposure concentration is 0~12.5 mg / L.

[0019] In some embodiments of the present invention, the sodium arsenite exposure concentrations are 0 mg / L, 6.25 mg / L, and 12.5 mg / L, respectively.

[0020] Preferably, the sodium arsenite exposure time is 7 to 21 days.

[0021] In some embodiments of the present invention, the sodium arsenite exposure time is 7 days, 14 days and 21 days, respectively.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) Based on the transcriptome expression data of the liver of the swamp frog after sodium arsenite exposure, the present invention screens candidate internal reference genes and combines real-time quantitative PCR verification and various stability evaluation methods to screen and obtain the SND1 and NDUFB2 internal reference gene combination suitable for quantitative analysis of liver genes of swamp frog under sodium arsenite exposure conditions; the internal reference gene combination can be used for standardized correction of target gene expression data under different sodium arsenite exposure concentrations and different exposure times.

[0024] (2) The expression stability of candidate internal reference genes was evaluated by ΔCt, geNorm, NormFinder and BestKeeper methods. The results showed that the combination of SND1 and NDUFB2 genes was stable in the liver samples of swamp frog under sodium arsenite exposure conditions and was suitable as an internal reference gene combination for target gene expression correction.

[0025] (3) The SND1 and NDUFB2 gene-specific primers provided by the present invention have good amplification specificity and amplification efficiency; the linear relationship of the standard curves and the amplification efficiency of the two primer pairs meet the requirements of real-time fluorescence quantitative PCR analysis, and the melting curves show a single amplification peak.

[0026] (4) The present invention uses the antioxidant-related gene SOD1 as the target gene to verify the standardization effect of the internal reference gene combination; the results show that using the geometric mean of the expression levels of SOD1 and NDUFB2 genes as the standardization factor can reduce the influence of the expression fluctuation of a single internal reference gene on the quantitative results of the target gene, and improve the accuracy, stability and reliability of the target gene expression analysis in the liver of the swamp frog.

[0027] (5) Therefore, the SND1 and NDUFB2 internal reference gene combination and its specific primers provided by the present invention can be used for real-time fluorescence quantitative PCR analysis of target genes in the liver of the swamp frog under sodium arsenite exposure conditions, providing a suitable standardized gene expression tool for molecular toxicology research on arsenic exposure in the swamp frog. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments are briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and do not constitute a limitation on the scope of protection of the present invention. For those skilled in the art, other related drawings can be obtained based on the following drawings without any creative effort.

[0029] Figure 1The melting curves of the amplification products of the 7 pairs of candidate internal reference gene primers in Example 2 are shown; where A: NDUFB2; B: SUCLG1; C: TOMM22; D: SND1; E: TUBA; F: GAPDH; G: ACTB, and the ordinate -d(RFU) / dT represents the first negative derivative of the fluorescence signal with temperature.

[0030] Figure 2 The Ct value distribution results of NDUFB2, SUCLG1, TOMM22, SND1, TUBA, GAPDH and ACTB under different sodium arsenite exposure conditions in Example 3 are shown in Figure 3. Among them, A is a box plot of Ct values ​​and B is an interquartile range analysis plot of Ct values.

[0031] Figure 3 The following graphs show the results of evaluating the expression stability of seven candidate internal reference genes using the geNorm method in Example 3. Among them, A is the analysis graph of all samples, B is the analysis graph of CK (control sample), C is the analysis graph of NaAsO2-L (low concentration sodium arsenite exposure), D is the analysis graph of NaAsO2-H (high concentration sodium arsenite exposure), E is the analysis graph of 7-day exposure time, F is the analysis graph of 14-day exposure time, and G is the analysis graph of 21-day exposure time.

[0032] Figure 4 In Example 3, the geNorm method was used to analyze the continuously standardized paired factor variation value V. n / V n+1 The results are shown in the figure; where A represents the paired variance analysis results for different exposure concentration groups, and B represents the paired variance analysis results for different exposure time groups.

[0033] Figure 5 The melting curve of the SOD1 gene primer amplification product in Example 4 is shown.

[0034] Figure 6 This is a graph showing the validation results of the standardization of the relative expression level of SOD1 using different internal reference genes in Example 4; where A, B, and C represent the detection results at 7d, 14d, and 21d after treatment, respectively; each graph uses SND1 as a single internal reference, a combination of SND1 and NDUFB2 as dual internal references, and GAPDH as a single internal reference for standardization, respectively; CK, NaAsO2-L, and NaAsO2-H represent the control group, the low-dose treatment group, and the high-dose treatment group, respectively.

[0035] Figure 7 The graph shows the results of SOD1 gene transcript abundance detection in different treatment groups 21 days after treatment in Example 4; CK, NaAsO2-L, and NaAsO2-H represent the control group, low-dose treatment group, and high-dose treatment group, respectively. Detailed Implementation

[0036] The technical solution of the present invention will be further described below with reference to specific embodiments. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Any equivalent substitutions, conventional adjustments, or improvements made based on the disclosure of the present invention without departing from the concept of the present invention should fall within the scope of protection of the present invention.

[0037] Unless otherwise specified, the terms used in the following implementation methods and embodiments generally have the meanings commonly understood by those skilled in the art.

[0038] Unless otherwise stated, the technical and scientific terms used in this specification have the meanings commonly understood by those skilled in the art. Experimental methods without specific conditions should be performed under standard conditions in the field, or as recommended by the manufacturers of the reagents and instruments used.

[0039] The terms "comprising," "including," and "having" used in this specification are open-ended expressions, meaning that in addition to the explicitly listed technical features, other technical features that do not affect the implementation of this invention may also be included.

[0040] The sodium arsenite exposure conditions described in this specification include different sodium arsenite exposure concentrations and / or different sodium arsenite exposure times. In a specific embodiment, three concentration conditions were set: sodium arsenite exposure concentrations of 0 mg / L, 6.25 mg / L, and 12.5 mg / L. 0 mg / L served as the control group (CK), 6.25 mg / L as the low-concentration sodium arsenite exposure group (NaAsO2-L), and 12.5 mg / L as the high-concentration sodium arsenite exposure group (NaAsO2-H). Sodium arsenite exposure times included 7 days, 14 days, and 21 days. These three concentration conditions combined with the three treatment times formed nine treatment conditions: 0 mg / L-7 days, 0 mg / L-14 days, 0 mg / L-21 days, 6.25 mg / L-7 days, 6.25 mg / L-14 days, 6.25 mg / L-21 days, 12.5 mg / L-7 days, 12.5 mg / L-14 days, and 12.5 mg / L-21 days.

[0041] In the evaluation of the stability of candidate internal control genes, experimental samples were stratified for different analytical purposes. The analytical datasets used in this specification, figures, and tables have the following meanings: "All Samples" represents the dataset including all experimental samples under nine treatment conditions; "CK" represents all control samples at 7d, 14d, and 21d under 0 mg / L sodium arsenite exposure; "NaAsO2-L" represents all samples at 7d, 14d, and 21d under 6.25 mg / L sodium arsenite exposure; "NaAsO2-H" represents all samples at 7d, 14d, and 21d under 12.5 mg / L sodium arsenite exposure; "7d," "14d," and "21d" represent analytical datasets divided by exposure time, where each time point includes the CK, NaAsO2-L, and NaAsO2-H samples at that time point. These analytical datasets are used to evaluate the expression stability of candidate internal control genes from different perspectives, including overall population, exposure concentration, and exposure time. There is sample overlap between the analytical datasets; they are not independent experimental treatment groups.

[0042] This invention uses SND1, NDUFB2, SUCLG1, TOMM22, TUBA, GAPDH, and ACTB as candidate internal reference genes. Among them, SND1, NDUFB2, SUCLG1, TOMM22, and TUBA were screened based on liver transcriptome expression data of the marsh frog under sodium arsenite exposure conditions, while GAPDH and ACTB were used as commonly used traditional internal reference genes for comparative analysis.

[0043] This invention employs four methods—ΔCt, geNorm, NormFinder, and BestKeeper—to evaluate the expression stability of candidate internal reference genes. The geometric mean is calculated based on the stability ranking of the candidate internal reference genes using these four methods, and the overall stability ranking of the candidate internal reference genes is obtained accordingly. A lower geometric mean indicates higher overall expression stability of the candidate internal reference gene.

[0044] In this specification, the relative expression level of the target gene is expressed in 22 -ΔΔCt The expression levels of target genes in the liver of the swamp frog were corrected using a standardization method. When using an internal reference gene combination consisting of the SND1 and NDUFB2 genes for standardization, the geometric mean of the expression levels of the SND1 and NDUFB2 genes was used as the standardization factor.

[0045] Example 1: Sodium arsenite exposure and preparation of liver samples from marsh frogs

[0046] The swamp frogs (Hylarana guentheri) used in the experiment were purchased from a breeding farm in Haikou and were acclimatized for one week at the animal experiment platform of the College of Life Sciences, Hainan Normal University. After the acclimatization period, healthy male swamp frogs of similar size were selected for the experiment, with an average weight of (30.0±8.1) g.

[0047] Sodium arsenite (NaAsO2, purity ≥90%) was purchased from Sigma-Aldrich. A 1 g / L stock solution of sodium arsenite was prepared using standard drinking water, and then diluted to the appropriate exposure concentration before the experiment.

[0048] To determine the concentrations for subsequent subchronic exposure experiments, sodium arsenite at concentrations of 6.25 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L, and 100 mg / L was used in a 96-hour acute toxicity pre-experiment with the marsh frog. The 96-hour median lethal concentration (LC50) of sodium arsenite for the marsh frog was determined. 50 The concentration was approximately 31.25 mg / L. Based on the results of the acute toxicity pre-test, 6.25 mg / L and 12.5 mg / L were determined as the concentrations for subsequent sodium arsenite exposure tests.

[0049] Swamp frogs were randomly divided into a control group, a low-concentration sodium arsenite exposure group, and a high-concentration sodium arsenite exposure group. The sodium arsenite concentration in the control group was 0 mg / L, in the low-concentration sodium arsenite exposure group it was 6.25 mg / L, and in the high-concentration sodium arsenite exposure group it was 12.5 mg / L. 12 L of exposure solution was added to each exposure container.

[0050] The exposure experiment lasted for 21 days, with liver samples collected on days 7, 14, and 21 of exposure. Six marsh frogs were randomly selected from each treatment group at each time point as biological replicates. The exposure solution was changed every 48 hours during the exposure period, with a 50% replacement rate each time, to maintain the target exposure concentration for each treatment group.

[0051] Prior to sampling, the marsh frogs were fasted to reduce the impact of dietary factors on liver gene expression. During the experiment, the activity status, feeding status, and mortality of the marsh frogs were observed daily, and abnormal behaviors were recorded.

[0052] After the appropriate exposure time was reached, the marsh frogs were anesthetized with urethane solution. Once the absence of pain reflexes was confirmed, they were humanely euthanized, and liver tissue was rapidly dissected. The obtained liver tissue was quickly rinsed with pre-cooled phosphate buffer, surface liquid was blotted off with sterile filter paper, aliquoted, and immediately flash-frozen in liquid nitrogen. It was then transferred to -80°C for storage and used for RNA extraction, transcriptome analysis, and real-time quantitative PCR detection.

[0053] Approximately 100 mg of liver tissue from the marsh frog was collected and thoroughly homogenized using a SCIENTZ-48 low-temperature high-throughput tissue homogenizer. Total RNA was extracted from the liver tissue according to the TRIzol® Reagent (Invitrogen, catalog number: 15596026CN) instructions and stored at -80°C for later use. The concentration and purity of total RNA were determined using a NanoDrop 2000 spectrophotometer.

[0054] Take 1 μg of qualified total RNA and use the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, catalog number: RR047A) for reverse transcription to synthesize cDNA. First, a genomic DNA removal reaction is performed in a 10 μL reaction system, including 1 μL of gDNA Eraser, 2 μL of 5×gDNA Eraser Buffer, and 1 μg of total RNA, brought to a final volume of 10 μL with RNase-Free dH2O. Then, a reverse transcription reaction is performed in a 20 μL reaction system, including 10 μL of the genomic DNA-removed reaction solution, 4 μL of 5×PrimeScript Buffer, 1 μL of PrimeScript RT Enzyme Mix 1, 1 μL of RT Primer Mix, and 4 μL of RNase-Free dH2O. The reverse transcription program is: 42 °C for 15 min, 95 °C for 3 min, and incubation at 4 °C. The cDNA obtained from reverse transcription is stored at -20 °C for later use.

[0055] Before real-time quantitative PCR detection, the obtained cDNA was diluted 5-fold with nuclease-free water. Three technical replicates were set up for each liver sample for subsequent expression detection of candidate internal control genes and target genes.

[0056] This experiment was reviewed and approved by the Experimental Animal Ethics Committee of the Hainan Provincial Ecological and Environmental Education Center of Hainan Normal University, with ethics approval number 2025-002.

[0057] Example 2: Candidate internal reference gene screening, primer design, and amplification performance evaluation

[0058] 1. Screening candidate internal reference genes based on transcriptome data

[0059] Transcriptome data from the livers of *Sphagnum mossula* after 21 days of sodium arsenite exposure were used for preliminary screening of candidate internal control genes. Transcriptome samples included a control group, a low-concentration sodium arsenite exposure group, and a high-concentration sodium arsenite exposure group, with three biological replicates in each group, totaling nine transcriptome samples. The transcriptome data were used to evaluate the expression stability of candidate genes among different treatment groups under the long-term sodium arsenite exposure endpoint.

[0060] Transcriptome sequencing libraries were constructed using qualified RNA samples. Gene expression matrices were obtained after high-throughput sequencing and quality control, and the relative expression abundance of each gene was expressed as TPM values.

[0061] Based on the gene expression matrix, the average relative expression level, intragroup coefficient of variation, and intergroup expression differences of each gene in the control group, low-concentration sodium arsenite exposure group, and high-concentration sodium arsenite exposure group were calculated.

[0062] The average TPM value greater than 10 was used as the screening criterion for expression abundance, and the maximum absolute log2FC less than 0.2 in pairwise comparisons between different treatment groups was used as the screening criterion for expression stability. Low-expression genes and genes significantly affected by sodium arsenite exposure were excluded.

[0063] For genes that meet the above screening criteria, calculate their within-group coefficient of variation in the control group, low-concentration sodium arsenite exposure group, and high-concentration sodium arsenite exposure group, and calculate the overall stability score according to the following formula:

[0064] Overall stability score = Max CV + max |log2FC|.

[0065] Where Max CV represents the maximum coefficient of variation within the three treatment groups, and max |log2FC| represents the maximum absolute log2FC in pairwise comparisons of the three treatment groups.

[0066] Based on the overall stability score, a total of 129 genes with relatively high expression abundance and small expression changes among treatment groups were selected. NDUFB2, SUCLG1, TOMM22, SND1, and TUBA were chosen as candidate internal reference genes from these, while traditional internal reference genes GAPDH and ACTB were included for comparison. The RNA-seq expression characteristics and overall stability scores of the seven candidate internal reference genes are shown in Table 1.

[0067] Table 1. RNA-seq expression characteristics and overall stability scores of candidate internal reference genes.

[0068]

[0069] The overall stability score represents the degree of fluctuation in gene expression levels. The lower the overall stability score, the more constant the expression level of the gene is under different samples and experimental conditions, and the more reliable and ideal it is as an internal reference.

[0070] Table 1 shows that NDUFB2 had the lowest overall stability score. Although SND1 did not have the most ideal overall stability score, its maximum absolute |log2FC| was 0.044, which was relatively small, indicating that the expression difference between the SND1 treatment groups was small. Both were superior to the traditional internal reference genes GAPDH and ACTB. Given that RNA-seq data only reflects the preliminary expression stability of candidate genes at a specific time point, further stability verification by real-time quantitative PCR was carried out.

[0071] It should be noted that the RNA-seq data listed in Table 1 only reflect the expression stability of candidate genes among different concentration treatment groups under the endpoint of 21 days of sodium arsenite exposure. The expression stability of candidate internal reference genes under different exposure times (7d, 14d, and 21d) and different sodium arsenite exposure concentrations was further verified by real-time quantitative PCR.

[0072] 2. Primer design for candidate internal reference genes

[0073] Based on the nucleotide sequences of SND1 (SEQ ID NO.1), NDUFB2 (SEQ ID NO.2), SUCLG1 (SEQ ID NO.3), TOMM22 (SEQ ID NO.4), TUBA (SEQ ID NO.5), GAPDH (SEQ ID NO.6), and ACTB (SEQ ID NO.7), specific real-time quantitative PCR primers were designed using Primer 5 software. These primers were synthesized by Sangon Biotech Co., Ltd.

[0074] Primers for amplifying the SND1, NDUFB2, SUCLG1, TOMM22, TUBA, GAPDH, and ACTB genes all include forward and reverse primers, with their nucleotide sequences shown in SEQ ID NO. 8 to SEQ ID NO. 21, respectively. The forward primer for amplifying SND1 has a nucleotide sequence of 5'-CCTGGTCATGGTGGAGGTTC-3', as shown in SEQ ID NO. 8; the reverse primer has a nucleotide sequence of 5'-AATCACCATAGCGCCAGAGG-3', as shown in SEQ ID NO. 9. Primer pairs for amplifying the NDUFB2 gene include forward and reverse primers. The forward primer has a nucleotide sequence of 5'-GATCCTGTGGCATTTGTGGC-3', as shown in SEQ ID NO. 10; the reverse primer has a nucleotide sequence of 5'-GCCCAGCTCTTCATCTGTCC-3', as shown in SEQ ID NO. 11.

[0075] 3. Evaluation of Real-Time Quantitative PCR Amplification Performance

[0076] Real-time quantitative PCR was performed using 2× SYBR Green qPCR Mix (No ROX) (GeneBetter, catalog number: P313-500) and a Roche LightCycler 480 II real-time quantitative PCR instrument. The 20 μL reaction volume consisted of 10 μL of 2× SYBR Green qPCR Mix (No ROX), 0.8 μL of forward primer, 0.8 μL of reverse primer, and 1 μL of cDNA template, brought to a final volume of 20 μL with nuclease-free water. The reaction program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing and extension for 30 s, for a total of 40 cycles. Melting curve analysis was performed after amplification. Three technical replicates were set up for each sample, and a template-free control was included.

[0077] Equal volumes of cDNA from liver samples of *Frogella maculatus* from each treatment group were mixed to prepare a mixed cDNA template, which was then serially diluted. Amplification was performed using the mixed cDNA at different dilutions as templates. A standard curve was established based on the template dilution factor and Ct value, and the amplification efficiency was calculated using the following formula:

[0078] E = (10 -1 / slope -1) ×100%

[0079] Where E represents amplification efficiency and slope represents the slope of the standard curve.

[0080] The primer sequences and amplification characteristics of the seven candidate internal reference genes are shown in Table 2. The amplification products of the seven pairs of candidate internal reference gene primers ranged in length from 89 bp to 132 bp, and the coefficient of determination R of the standard curve was [missing information]. 2 The values ​​ranged from 0.9954 to 0.9999, and the amplification efficiency ranged from 90.86% to 103.01%. This indicates that the designed primers meet the requirements for real-time quantitative PCR analysis.

[0081] Table 2 Primer sequences and amplification characteristics of candidate internal reference genes for real-time quantitative PCR

[0082]

[0083] Melting curves of the amplification products of 7 pairs of candidate internal reference gene primers are shown below. Figure 1 As shown, all seven pairs of candidate internal reference gene primers exhibited a single melting peak, and no obvious non-specific amplification peaks or primer dimer peaks were observed, indicating that the designed primers have good amplification specificity.

[0084] Example 3 Evaluation of expression stability of candidate internal reference genes

[0085] 1. Distribution of Ct values ​​of candidate internal reference genes

[0086] Specific primers for seven candidate internal reference genes (SND1, NDUFB2, SUCLG1, TOMM22, TUBA, GAPDH, and ACTB) were used to perform real-time quantitative PCR detection on cDNA samples from the livers of *Rhizophora stylosa* obtained under different treatment conditions. Three concentration conditions were set up: 0 mg / L, 6.25 mg / L, and 12.5 mg / L. 0 mg / L served as the control condition, while 6.25 mg / L and 12.5 mg / L represented low and high concentrations of sodium arsenite exposure, respectively. Treatment times were 7 days, 14 days, and 21 days, resulting in nine treatment conditions: 0 mg / L-7d, 0 mg / L-14d, 0 mg / L-21d, 6.25 mg / L-7d, 6.25 mg / L-14d, 6.25 mg / L-21d, 12.5 mg / L-7d, 12.5 mg / L-14d, and 12.5 mg / L-21d. Liver cDNA samples obtained under the above treatment conditions were analyzed by real-time quantitative PCR to determine the Ct values ​​of each candidate internal control gene. The obtained Ct value data were further stratified according to the analytical purpose, by all samples, concentration conditions, and treatment time, for the evaluation of the expression stability of the candidate internal control genes.

[0087] like Figure 2 As shown in Figure A, the Ct values ​​of the seven candidate internal reference genes are generally distributed between approximately 19 and 35, all of which can be effectively detected. Figure 2As shown in Figure B, the interquartile ranges of the Ct values ​​for SND1 and NDUFB2 are 2.56 and 2.67, respectively, indicating that the Ct values ​​of SND1 and NDUFB2 have relatively low dispersion. Since the dispersion of Ct values ​​cannot alone reflect the overall stability of the internal reference genes, further evaluation of the applicability of SND1 and NDUFB2 as internal reference genes for quantitative real-time PCR will be conducted by combining various stability evaluation algorithms and target gene expression verification.

[0088] 2. Comparative stability analysis of ΔCt

[0089] The mean standard deviation of candidate internal reference genes was calculated using the comparison ΔCt method. The smaller the value, the higher the expression stability. The results are shown in Table 3.

[0090] Table 3. Ranking results of candidate internal reference gene expression stability evaluated by the ΔCt method in different analysis datasets. .

[0091] Note: In the table, "Gene / Value" represents the name of the candidate internal control gene and its mean standard deviation using the ΔCt method, respectively; the smaller the value, the higher the expression stability of the candidate internal control gene. All sample groups represent the dataset of all experimental samples under 9 treatment conditions; CK represents all control samples at 7d, 14d, and 21d under 0 mg / L condition; NaAsO2-L represents all samples at 7d, 14d, and 21d under 6.25 mg / L sodium arsenite exposure condition; NaAsO2-H represents all samples at 7d, 14d, and 21d under 12.5 mg / L sodium arsenite exposure condition; 7d, 14d, and 21d represent the datasets at the corresponding time points, each time point including CK, NaAsO2-L, and NaAsO2-H samples.

[0092] Table 3 shows that in all sample groups, the mean standard deviations of SND1 and NDUFB2 were 1.543 and 1.580, respectively, ranking first and second in stability. NDUFB2 ranked first in the CK, 7d, and 14d group datasets. These results indicate that SND1 and NDUFB2 are stably expressed in multiple analytical datasets and can be considered preferred internal reference genes for real-time quantitative PCR of the liver of the water frog.

[0093] 3. Stability and paired variation analysis of geNorm

[0094] The expression stability value M of the seven candidate internal reference genes was calculated using the geNorm method. A smaller M value indicates higher expression stability. Results are shown in [Figure number missing]. Figure 3SND1 showed high stability in all samples, CK, NaAsO2-L, and 14d analysis datasets; NDUFB2 performed well in CK, NaAsO2-H, and 14d analysis datasets, indicating that the two have certain stability and complementarity under different sodium arsenite exposure conditions.

[0095] Further calculate the paired variation value V n / V n+1 The results are shown Figure 4 In the analysis of the datasets for all sample groups, the control group (CK), the low concentration group (NaAsO2-L), and the high concentration group (NaAsO2-H), the lowest paired variance values ​​were 0.252, 0.269, 0.177, and 0.212, respectively; in the analysis of the 7d, 14d, and 21d datasets, the lowest paired variance values ​​were 0.231, 0.272, and 0.213, respectively, all higher than 0.15. This indicates that the optimal number of internal reference genes is at least two.

[0096] Therefore, the geNorm results support that SND1 and NDUFB2 have good expression stability and certain complementarity under different treatment conditions, but the combination of internal reference genes still needs to be determined by comparing ΔCt, NormFinder, BestKeeper, comprehensive stability ranking and SOD1 target gene verification results.

[0097] 4. Stability analysis of NormFinder and BestKeeper

[0098] The NormFinder method was used to calculate stability values ​​based on the intra- and inter-group expression variations of candidate internal reference genes. Lower stability values ​​indicate higher expression stability of the candidate internal reference genes; the results are shown in Table 4.

[0099] Table 4. Ranking results of candidate internal reference gene expression stability evaluated by the NormFinder method in different analysis datasets. .

[0100] Note: In the table, "Gene / Value" represents the name of the candidate internal control gene and its NormFinder stability value, respectively; the smaller the value, the higher the expression stability of the candidate internal control gene; "All Samples" represents the dataset of all experimental samples under 9 treatment conditions; "CK" represents all control samples at 7d, 14d, and 21d under 0 mg / L condition; "NaAsO2-L" represents all samples at 7d, 14d, and 21d under 6.25 mg / L sodium arsenite exposure condition; "NaAsO2-H" represents all samples at 7d, 14d, and 21d under 12.5 mg / L sodium arsenite exposure condition; "7d", "14d", and "21d" represent the datasets at the corresponding time points, and each time point includes CK, NaAsO2-L, and NaAsO2-H samples.

[0101] The overall analysis results of all samples show that NDUFB2 ranks first in stability, SND1 ranks second, and GAPDH has relatively low stability.

[0102] The standard deviation (SD), coefficient of variation (CV), and correlation coefficient (r) of the Ct values ​​of candidate internal reference genes were calculated using the BestKeeper method, and the three indicators were ranked. SD and CV were ranked from smallest to largest, and r was ranked from largest to smallest. The geometric mean of each indicator was calculated as the BestKeeper overall stability ranking value. The formula for calculating the geometric mean of the BestKeeper stability ranking is as follows: ; in, , and The values ​​represent the ranking of candidate reference genes in terms of SD, CV, and correlation coefficient r, respectively. The smaller the G value, the higher the overall stability ranking of the candidate reference gene. The results are shown in Table 5.

[0103] Table 5. Ranking results of candidate internal reference gene expression stability evaluated by the BestKeeper method in different analysis datasets. .

[0104] Note: In the table, "Gene / Value" represents the candidate internal control gene name and its BestKeeper stability ranking geometric mean, respectively; the smaller the value, the higher the expression stability of the candidate internal control gene; "All Samples" represents the dataset of all experimental samples under 9 treatment conditions; "CK" represents all control samples at 7d, 14d, and 21d under 0 mg / L condition; "NaAsO2-L" represents all samples at 7d, 14d, and 21d under 6.25 mg / L sodium arsenite exposure condition; "NaAsO2-H" represents all samples at 7d, 14d, and 21d under 12.5 mg / L sodium arsenite exposure condition; "7d", "14d", and "21d" represent the datasets at the corresponding time points, and each time point includes CK, NaAsO2-L, and NaAsO2-H samples.

[0105] In the entire sample dataset, TOMM22, NDUFB2, and SND1 ranked relatively high in BestKeeper's overall evaluation, while GAPDH ranked relatively low.

[0106] 5. Overall Stability Ranking

[0107] To reduce bias caused by evaluation using a single algorithm, the stability rankings of the seven candidate internal reference genes were obtained in comparative analyses using ΔCt, geNorm, NormFinder, and BestKeeper. Furthermore, the geometric mean of the analysis results from the four algorithms was calculated using the comprehensive analysis tool RefFinder. A smaller geometric mean indicates higher overall stability of the candidate internal reference genes. The overall stability ranking results under different sodium arsenite treatment conditions are shown in Table 6.

[0108] Table 6. Overall stability ranking of candidate internal reference gene expression stability .

[0109] Note: In the table, "Gene / Value" represents the candidate internal control gene name and its overall stability ranking geometric mean, respectively; the smaller the value, the higher the overall expression stability of the candidate internal control gene; "All Samples" represents the dataset of all experimental samples under 9 treatment conditions; "CK" represents all control samples at 7d, 14d, and 21d under 0 mg / L condition; "NaAsO2-L" represents all samples at 7d, 14d, and 21d under 6.25 mg / L sodium arsenite exposure condition; "NaAsO2-H" represents all samples at 7d, 14d, and 21d under 12.5 mg / L sodium arsenite exposure condition; "7d", "14d", and "21d" represent the datasets at the corresponding time points, and each time point includes CK, NaAsO2-L, and NaAsO2-H samples.

[0110] Based on the comprehensive analysis of all samples, SND1 ranked first in overall stability with a geometric mean of 1.732; NDUFB2 ranked second with a mean of 1.861; and GAPDH ranked relatively low in overall stability with a mean of 6.481. Combining the four stability evaluation methods and the overall stability ranking results, SND1 and NDUFB2 both exhibited high overall expression stability under different treatment conditions.

[0111] Therefore, this invention selects SND1 and NDUFB2 as the internal reference gene combination for real-time quantitative PCR of the liver of the swamp frog, and uses the geometric mean of their expression levels as a standardization factor for the standardization correction of the target gene expression level under sodium arsenite exposure conditions.

[0112] Example 4: Application Verification of the SND1 and NDUFB2 Internal Reference Gene Combination

[0113] To verify the applicability of SND1 and NDUFB2 as internal reference genes in real-time quantitative PCR of the liver of the marsh frog under sodium arsenite exposure conditions, SOD1 (nucleotide sequence shown in SEQ ID NO. 22) was selected as the target gene for relative expression analysis. The forward primer for SOD1 was 5'-GCAGGAAGGAAGTGGACCTG-3' (sequence shown in SEQ ID NO. 23), and the reverse primer was 5'-AGGTCTCCAACATGCCTCAC-3' (sequence shown in SEQ ID NO. 24), with an amplification product length of 186 bp. The standard curve of this primer showed a coefficient of determination R² of 0.9992, an amplification efficiency of 102.01%, and a single peak in the melting curve, indicating good amplification specificity and efficiency. Figure 5 ).

[0114] The relative expression level of the SOD1 gene was used as 2 -ΔΔCt The expression levels were calculated using a calibrated method. The corresponding control group was used as the calibration group at each time point. Normalization was performed using SND1 as a single internal control, the SND1 / NDUFB2 dual internal control combination, and the GAPDH single internal control, respectively; for the dual internal control combination, the geometric mean of SND1 and NDUFB2 expression levels was used as the normalization factor. Three biological replicates were set up for each group, based on 2... -ΔΔCt Two-way ANOVA was performed on the values, and Tukey's method was used for multiple comparisons. The results are shown in [Figure number missing]. Figure 6 .

[0115] The results are as follows Figure 6 As shown, the stable internal reference gene SND1, the dual internal reference gene combination SND1 and NDUFB2, and the internal reference gene GAPDH with poor stability were used as standardized internal references to detect the relative expression level of superoxide dismutase 1 gene SOD1 under different exposure concentrations and exposure times.

[0116] The results showed that, when SND1 was used as a single internal reference for standardization, at 7 days of exposure, the relative expression level of SOD1 was higher in the CK group and relatively lower in the NaAsO2-L and NaAsO2-H groups; at 14 days of exposure, the relative expression level of SOD1 increased in NaAsO2-L and relatively lower in NaAsO2-H; at 21 days of exposure, the relative expression level of SOD1 was significantly increased in NaAsO2-H, followed by NaAsO2-L, and relatively lower in CK.

[0117] The relative expression level of SOD1, obtained by normalization using the SND1 and NDUFB2 dual internal reference combination, showed a similar trend to that of the SND1 single internal reference: at 7 days of exposure, the relative expression level of SOD1 was higher in the CK group and relatively lower in the NaAsO2-L and NaAsO2-H groups; at 14 days of exposure, the relative expression level of SOD1 increased in the NaAsO2-L group and was lower in the NaAsO2-H group; at 21 days of exposure, the relative expression level of SOD1 was significantly higher in the NaAsO2-H group, followed by the NaAsO2-L group, and relatively lower in the CK group.

[0118] When GAPDH was used as an internal reference gene for standardization, the trend of the relative expression level of SOD1 was significantly different from that obtained by using SND1 as a single internal reference or a combination of SND1 and NDUFB2 as dual internal references, especially showing different expression patterns at 7d and 14d of exposure.

[0119] To further verify the reliability of the relative SOD1 expression levels obtained from different internal reference genes, the transcript abundance of the SOD1 gene was analyzed based on the liver transcriptome data of the CK group, NaAsO2-L group, and NaAsO2-H group after 21 days of exposure. The results are shown in [Figure number missing]. Figure 7 .

[0120] from Figure 7 As can be seen, after 21 days of exposure, the abundance of SOD1 gene transcripts increased with increasing NaAsO2 exposure concentration, with the highest abundance in the NaAsO2-H group, followed by the NaAsO2-L group, and the lowest abundance in the CK group. This trend is consistent with... Figure 6 The relative expression trends of SOD1 obtained by standardization using SND1 as a single internal reference and the combination of SND1 and NDUFB2 as dual internal references in Figure C are consistent.

[0121] The above results indicate that the stability of the internal reference gene can affect the analysis results of the relative expression level of the target gene. The transcriptome sequencing results further show that the SND1 and NDUFB2 gene combination obtained by screening has good expression stability and is suitable for the standardized analysis of the relative expression level of the SOD1 gene under the exposure conditions described in this embodiment.

[0122] The above embodiments are only used to illustrate preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Equivalent substitutions, variations, or improvements made by those skilled in the art without departing from the concept of the present invention should all fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of SND1 and NDUFB2 dual internal reference genes in real-time quantitative PCR of liver tissue from the marsh frog, characterized in that, The application of SND1 and NDUFB2 as dual internal reference genes in the detection of target gene expression in the liver tissue of the swamp frog under sodium arsenite exposure conditions using real-time PCR. The nucleotide sequence of SND1 is shown in SEQ ID NO.1, and the nucleotide sequence of NDUFB2 is shown in SEQ ID NO.

2. The target gene in the liver tissue of the swamp frog is the superoxide dismutase 1 gene.

2. The application according to claim 1, characterized in that, The primers for amplifying SND1 include the forward primer shown in SEQ ID NO.8 and the reverse primer shown in SEQ ID NO.9; the primers for amplifying NDUFB2 include the forward primer shown in SEQ ID NO.10 and the reverse primer shown in SEQ ID NO.

11.

3. The application according to claim 1, characterized in that, The sodium arsenite exposure conditions include the sodium arsenite exposure concentration and the sodium arsenite exposure time.

4. The application according to claim 3, characterized in that, The sodium arsenite exposure concentration is 0~12.5 mg / L; the sodium arsenite exposure time is 7~21 days.

Images