S1000tm thermal cycler

Total RNA was extracted from human placentas and HTR8 cell lines using a TRIzol kit (Invitrogen, USA). First-strand cDNA was synthesized to a final volume of 20 µl using a SuperScript RIII first-strand kit (Invitrogen, USA). Following reverse transcription, PCR amplification of the cDNA was performed using the corresponding specific primers (note: the sequences of the primers are provided in Supplementary file 1: Table S2). Primers were obtained from Sangon Biotech (Shanghai, China). q-PCR was performed using 2× RealStar Fast SYBR qPCR Mix (A304-10, GenStar, China) in a Bio-Rad S1000TM Thermal cycler (Bio-Rad, USA). The reaction conditions were as follows: 95 °C for 2 min, 40 cycles at 95 °C for 15 s and 60 °C for 30 s, melt curve 65 to 95 °C, increment 0.5 for 5 s. The target gene mRNA expression levels were normalized using 18 S as a reference. The results were analyzed using the 2^−ΔΔCt method.

Total RNA was extracted from human placentas and HTR8 cell lines using a TRIzol kit (Invitrogen, USA). First-strand cDNA was synthesized to a final volume of 20 µl using a SuperScript RIII first-strand kit (Invitrogen, USA). Following reverse transcription, PCR amplification of the cDNA was performed using the corresponding specific primers (note: the sequences of the primers are provided in Supplementary file 1: Table S2). Primers were obtained from Sangon Biotech (Shanghai, China). q-PCR was performed using 2× RealStar Fast SYBR qPCR Mix (A304-10, GenStar, China) in a Bio-Rad S1000TM Thermal cycler (Bio-Rad, USA). The reaction conditions were as follows: 95 °C for 2 min, 40 cycles at 95 °C for 15 s and 60 °C for 30 s, melt curve 65 to 95 °C, increment 0.5 for 5 s. The target gene mRNA expression levels were normalized using 18 S as a reference. The results were analyzed using the 2^−ΔΔCt method.

For cloning the mango MiSOC1 gene, we first isolated partial mango MiSOC1 cDNA fragment with degenerate PCR (polymerase chain action) method, then obtain the full length mango MiSOC1 cDNA sequence with 3′and 5′ RACE. The primer sets are listed in Table 1 designed based on the 5′ and 3′-terminal sequence of partial mango MiSOC1 sequence obtained in our laboratory. The reaction was performed in an S1000TM thermal cycler (Bio-Rad, USA). The sequencing results of the 3′ and 5′ RACE PCR products were searched against the NCBI database, then determined the splice and clone the full length of MiSOC1gene. The 3′ and 5′ RACE PCR were performed with the protocol of the 3′ and 5′ full RACE Core Set (TaKaRa, Japan). The PCR production was purified using a PCR purification kit (Qiagen), inserted into the pGEM-T Easy Vector (Promega) and transformed into competent Escherichia coli DH5a cell. The recombinant plasmids were identified and the positive clones were picked and sequenced in Shanghai Sangon Biological Engineering Technology and Services Co., Ltd (Shanghai China). The confirmed full-length cDNA of MiSOC1was was deposited at GenBank (accession number KP404094) and used for molecular characterization and bioinformatics analysis later.

Frozen muscle (10 to 15 mg) was used to isolate RNA using the RNeasy Mini Kit (Qiagen, Canada) according to the manufacturer’s instructions. Samples were homogenised using the TissueLyser II (Qiagen, Canada). RNA concentration and purity was determined spectrophotometrically (NanoDrop 2000, Thermo Fisher Scientific, Wilmington, DE). For each sample, 1 μg of RNA was transcribed into cDNA on a thermal cycler (S1000TM Thermal Cycler, Bio-Rad, USA) using the iScriptTM cDNA Synthesis Kit (Bio-Rad, USA) and the following incubation profile: 5 min at 25 °C, 30 min at 42 °C, and 5 min at 85 °C. The transcription was performed with random hexamers and oligo dTs in accordance with the manufacturer’s instructions. A reverse transcriptase (RT)-negative control was also generated. cDNA was stored at −20°C until subsequent analysis.

MiRs were extracted from plasma using a miRcute miRNA isolation kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions. cDNAs were synthesized using a miRcute miRNA First-Strand cDNA Synthesis kit (Tiangen) in an S1000TM Thermal Cycler (Bio-Rad, USA). The miRcute miRNA qPCR detection kit (SYBR Green) (Tiangen) was used to assess the expression of individual miRs. The primers used were purchased from Tiangen Biotech Co., Ltd., catalog numbers were CD201-0114 (hsa-miR-378), CD201-0003 (hsa-miR-1), CD202-0006 (mmu-miR-133a) and CD201-0145 (hsa-U6). PCR amplification was performed using a C1000TM Thermal Cycler (CFX96TM Rea-Time System, Bio-Rad, USA) under the following conditions: 94°C for 2 min followed by 45 cycles of 94°C for 20 sec and 60°C for 34 sec. The production of specific products was confirmed using melting curve analysis (60-95°C) at the end of the amplification cycle. All experiments were performed in triplicate for each miRNA to obtain Ct values, and a non-template control was included on the same plate. A small stably expressed housekeeping RNA molecule, hsa-U6, was included as an internal control to normalize gene expression levels. Data were analyzed using Bio-Rad CFX Manager software (Bio-Rad), and the relative expression (fold difference) of candidate genes was calculated using the 2^−ΔΔCt method.