Specific primers

DNA was extracted using QIAamp DSP DNA Blood Mini Kit (QIAGEN, USA) and was stored in a refrigerator at −20 °C. The PCR amplification was performed using specific primers (Macrogen, Seoul, South Korea). The primer sequences are as follows: TUG1 rs5749201 F: 5′-TGC CTG CAT TTA CTG TCT TTG C-3′, R: 5′-TGT TGT GGT GTA TGT GGG CA-3′; TUG1 rs886471 F: 5′-ATG TCT AGG CTG TGT GGT TGG-3′, R: 5′-GAG CCC GCT TGC TAA AAG TC-3′, with a total volume of 25 μL including the following: 12.5 µL of a ready to use master mix (Promega, Woods Hollow Road Madison, WI, USA), 1.5 µL of each primer (0.5 µmol), 5.5 µL of template DNA (100–500 ng) and 1.0 µL of sterile deionized water. The thermal cycler profile (Biometra T professional thermocycler 070-851, Germany)). The cycling conditions included an initial denaturation step at 95 °C for 5 min followed by 35 cycles at 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s and a final extension of 7 min at 72 °C. After PCR amplification, the products were purified and then subjected to Sanger sequencing. By comparing the sequencing results to the sequencing data in the dbSNP database, the genotypes of TUG1 rs5749201 and rs886471 were determined.

E. tarda was detected by amplification of ABR genes (qnrA, blaTEM, and sul3) and gyrB gene by PCR (Bio-Rad, USA) using E. tarda-specific primers (Macrogen, Korea). A total of 25 μl of PCR reaction solution containing 1 μl of template DNA, 1 μl forward primer, 1 μl reverse primer, 10 μl injection water, and 12 μl GoTaq Green Master Mix (Promega, USA) were used to detect the ABR genes of E. tarda under amplification conditions for PCR as mentioned in Table 1. Amplified PCR products were analyzed on 1% agarose gel stained with ethidium bromide (C₂₁H₂₀BrN₃) and utilizing a standard-sized molecular marker (1Kb DNA Ladder RTU, GeneDireX). PCR products revealing the thickest bands were sequenced by Sanger’s method at BGI Hong Kong Co. Ltd., China.

At least 20 blastocysts from each group (control and NT-4-treated group) were collected in 1.5 mL microcentrifuge tubes (SPL Life Sciences, Co. Ltd., Pocheon, Gyeonggi-do, Republic of Korea), washed with DPBS and stored at −80 °C until analysis. Total RNA was extracted from blastocysts using the TRIzol reagent (TaKaRa Bio, Inc., Otsu, Shiga, Japan). cDNA synthesis was performed using a 5× reverse transcription master mixture (Elpis Bio, Inc., Chungcheongnam-do, Daejeon, Republic of Korea) following the manufacturer’s protocol. To perform qRT-PCR, the synthesized cDNA (0.5 μg/μL) was mixed with 2× SYBR Premix Ex Taq (TaKaRa Bio Inc.) and 10 pmol of specific primers (Macrogen, Inc., Seoul, Republic of Korea). The primers used in this experiment are listed in Supplementary Table S1. The qRT-PCR analysis was carried out using a CFX96 Touch real-time PCR detection system (Bio-Rad, Hercules, CA, United States), and the reactions were performed as follows: pre-denaturation at 95 °C for 5 min and 40 cycles of denaturation at 95 °C for 15°s, annealing at 57 °C for 15°s, and extension at 72 °C for 30 s. Data were collected at the extension phase of each cycle, and the relative quantification (R) value was calculated using the following equation: R = 2^{-[ΔCtsample−ΔCtcontrol]}. Normalization of gene expression levels was performed using the 18S ribosomal RNA (RN18S) gene as the control.

“Favor Prep Blood Genomic DNA Extraction Kit (Biotech Corp, Taiwan China)” was used for extracting DNA. Polymerase chain reaction (PCR) was employed to amplify DNA using specific primers (Macrogen Inc Korea) designed against the polymorphic cytosine–adenine repeat 1kb upstream of the human IGF-1 gene, i.e., sense: 5’-ACCACTCTGGGAGAAGGGTA-3, antisense: 5-GCTAGCCAGCTGGTGTTATT-3’. PCR amplification reactions were carried out in a 25μL reaction mixture which contained, 1μl (50 ng) of genomic DNA, 0.5 μl of each primer (5pM), 12.5 μl of 2x PCR master mix and 10.5 μl double distilled water. Amplification was performed on BioRad thermal cycler (iCycler, BioRad USA). The cycling conditions for the PCR were a cycle of initial denaturation at 94ºC for five minutes, followed by 35 cycles with denaturation for one minute at 94ºC, annealing at 58ºC for 60 seconds, extension at 72ºC for 60 sec, and with a final extension for 10 minutes. at 72ºC. The amplicons were run for 45 minutes on 3% agarose gel. The PCR product was checked under ultraviolet gel documentation system (Bio-Rad USA) and results were recorded.
IGF-1 levels in serum samples of both RA group and healthy group was measured by Enzyme Immunoassay kit (Bioassay technology Laboratory Shanghai China) following manufacturer’s instructions on automated ELISA CODA (BioRad, USA).