This study was approved by the Institutional Animal Care and Use Committee of the National Institutes For Food And Drug Control(IACUC permit number:2022(B)025). All research was performed in accordance with relevant guidelines and regulations.
1 Animal Sample
Two outbred groups of experimental rat were used in this research: SD and Wistar, 24 samples per strain, 7-8 weeks of age, half-sex.12 most commonly used inbred strains were selected for SNP determination: BN/SsNSlcNifdc ,F344/NSlcNifdc,DA,PVG,Lewis,SHR/NCrl,WKY/NCrl,UH rat model,CHN1,CHN2,CHN5,F344RGTM.6 samples of each strain,7-8 weeks old.
BN/SsNSlcNifdc and F344/NSlcNifdc were from National Rodent Laboratory Animal Resources Center,the production license was SCXK(Jing)2022-0002.DA,PVG,Lewis were from the Shanghai Institute for Biomedical and Pharmaceutical Technologies, the production license was SCXK(hu)2018-0006.SHR/NCrl,WKY/NCrl were from Charles River Laboratories,the production license was SCXK (Jing)2021-0006.The umbilical hernia (UH) rat model was provided by the Laboratory Animal Center, Academy of Military Medical Sciences. CHN1,CHN2,CHN5 were from Shaanxi Academy of Traditional Chinese Medicine and F344RGTM were from VITALSTAR.
Place the rats in a euthanasia box (note: rats cannot be stacked in containers) and introduce carbon dioxide; In the induction phase first, air or oxygen was provided, and then the CO2 concentration was continuously increased, and the rats were observed to determine that there was no breathing and no heartbeat, and the corpse was removed. All the samples are the tails of rat. Cut the tail of the mouse about 0.2cm~0.5cm, and put it to -20°C refrigerator for storage.
All animal procedures were based on 3R principle and approved by the Institutional Animal Care and Use Committee of the National Institutes For Food And Drug Control 2022(B)025.
2 DNA Extraction
Phenol-chloroform extraction method was used to extract DNA from tails[11].DNA samples were quantified spectrophotometrically by measuring absorbance at 260 nm, diluted to 50 ng/µL, and stored at 4°C.
3 SNPs Locus Selection
By searching PubMed and using ensemble database to analyze animal genetic information,SNPs sites for further screening were obtained[12][13].Then,the SNP loci suitable for amplification in inbred and outbred rats were screened by generation sequencing and DNA hybrid pool construction methods.
4 Oligonucleotides
Mmultiplex PCR primers were designed by Primer Premier 6 and ASPE primer were designed by Oligo 7.0 (Molecular Biology Insights, Vondelpark Colorado Springs, CO 80907). The ASPE primer contains two parts, the 5 'end is connected to the unique TAGs (specific 24-mer oligonucleotide sequences that complements to the anti-TAGs on xTAG beads), and the 3' nucleotide is the base of the SNP variant.All oligonucleotides were synthesized by Sangon Biotech, that are unmodified and purified by PAGE.
5 Multiplex PCR
Five separate multiplex PCR panels were required to generate all the fragments containing the 38 SNPs. The PCR mixtures contained 3uL of 10×PCR buffer, 2.4uL of dNTP,1U of HotStarTaq ® polymerase(Takara), varying concentrations of primers (Table 1) and 50 ng of genomic DNA in a final volume of 30 µL.Following an initial pre-denaturation step at 95°C for 5 min, the reactions were cycled 30 times through denaturation at 94°C for 30s, annealing temperatures for 30s and extension at 72°C for 45s.The reactions were then held at 4°C. The PCR products were subjected to 2.0% agarose gel electrophoresis to verify successful amplification of the desired fragments.
6 PCR product purification
First Exo-SAP compound enzyme mixture, which contains 1uL exonucleaseI, 6.25 uL Alkaline Phosphatase (Shrimp), 2.75 uLddH2O.Then, 10 µL of each PCR products was treated with1µL Exo/SAP enzyme mixture by incubating at 37°C for 30 min, folowed by enzyme deactivation at 80°C for 15min.
7 Allele-specific primer extension(ASPE)
Multiplex ASPE reactions comprised 2.5µL of 10×PCR buffer, 0.25µL biotin-dCTP(400µM ),0.15 µL HotStarTaq ® polymerase(5U/µL),100 µM each of dATP, dGTP, and dTTP, 1 µL of TAG-ASPE primer mix (500 nM each),10.1µL of ddH2O and 5 µL Target Exo-SAP treated PCR products. The ASPE primer sequences are listed in Table 2. The ASPE reactions were incubated at 96°C for 2 min and then subjected to 30 cycles at 94°C for30 s, 55°C for 1 min, and 72°C for 2 min. The samples were kept at 4°C until use.
8 Hybridization and detection
The target microspheres in each panel were mixed and then diluted to 100 of microspheres set per uL in 1× hybridization buffer[0.2 M NaCl, 0.1 M Tris (pH 8.0), 0.08% Triton X-100] .Aliquot 22.5 μL of the MagPlex-TAG microsphere mixture to each well.Add 2.5 μL of ddH2O to each background well.Add 2.5 μL of each sample to the appropriate wells.Then react at 96 ? for 90 s and 37 ? for 30min a thermal cycler.Prepare reporter mix by diluting SAPE to 10 μg/mL in 1X Tm hybridization buffer containing 0.1% BSA.Add 100 μL reporter mix to each well.Mix gently with pipette.Incubate at 37°C for 15 minutes and then analyze 100 μL at 37°C on the Luminex 200 System(HHA430).
9 Method accuracy verification
BN, F344, and SD samples were selected to verify the genotyping of 38 SNP sites by generation sequencing, and compared with Luminex-SNP genotyping method.
10 Data analysis
10.1 SNP typing data analysis
The data were acquired using the xPONENT3.1 system and analyzed using the Microsoft Excel.We determined the genotypes according to the median fluorescence intensity (MFI).The calculation formula is as follows:
In general,when the ratio was <0.25, the genotype was homozygous mutant type,when the ratio was 0.25-0.75,the genotype was heterozygous,when the ratio was >0.75,the genotype was homozygous wild type.
10.2 The inbred rat data
The inbred rat data only need to obtain the results of SNP genotyping .
10.3 The outbred rat data
We selected 23 SNP loci with good polymorphisms in both SD and WIistar populations for Popgene32 software analysis,and calculated the site Effective number of alleles(Ne),Shannon's Information index(I),observed heterozygosity (Ho), expected heterozygosity (He),Fis index.SNP loci were tested for polymorphism information content( PIC) by PIC-Calc 0.6[14].