Itaq universal sybr green supermix

Total RNA was isolated immediately after dissection using a Quick RNA isolation Kit (Huayueyang Biotechnology (Beijing) Co., LTD., Beijing, China). The reactions were performed using iTaq^TM Universal SYBR^® Green Supermix (BioRad, Hercules, CA, USA) in a 20 μl volume containing 10 μl of iTaq^TM Universal SYBR^® Green Supermix (2 × ), 0.4 μM each specific forward and reverse primer. 2 μl of 10-fold diluted template was used for 50 μl PCR reaction using primers listed in Table S2. The qRT-PCR was carried out in a Roche LightCycle 480 (Roche Applied Science, Mannheim, Germany) with one cycle of 95 °C 30 s, 40 cycles of 95 °C 5 s, 60 °C 30 s. A melt curve was performed at the end of each reaction to verify PCR product specificity. The 2^−△△Ct method was used to calculate the relative expression level51 (link). Beta-actin was used as an internal refs 39 (link), 40 , 41 (link). Changes in mRNA level of the test gene for each treatment were normalized to that of beta-actin.

Quantitative Real Time PCR (qRT-PCR) was used to determine the relative quantities of fs800 transcribed in fs800 RNAi treated worm pairs from 20, 29, 36 and 45 dpi. Three μL of cDNA was used for relative quantification with gene specific primers and iTaq^™ Universal SYBR^® Green Supermix (BioRad) containing hot-start iTaq DNA polymerase, dNTPs, MgCl2, SYBR^® Green I dye, and ROX reference dye. Primers were designed using Primer-Blast tool by NCBI, forward primer 5^′-CTCGTGGAAATAGTGCAAAAGG-3 ‘and reverse primer 5^′-GTCGACCA-TATCTATCTGTTCC-3’. GAPDH (XM_018794048.1) was used as an endogenous control for knockdown groups and controls. The qRT-PCR reaction was performed in 10 μL reaction and contained 5 μl iTaq Universal SYBR^® Green Supermix (BioRad), 3 μl cDNA (50 μg/μl), 1 μl each of forward 5′- GTG AAA GAG ATC CAG CAA ACA T −3′ and reverse 5′- ATA TGA GCC TGA GCT TTA TCA ATG-3′ primers 10 μM. The qRT-PCR profile was 55 °C for 2 min, 98 °C for 10 min, 40 cycles of 98 °C for 15 s, 60 °C for 1 min, and a final step of 60 °C for 5 min (Applied Biosystems 7500 FastReal-Time PCR System).
Comparative fs800 expression between S. mansoni knockdown group and control was calculated based on qRT-PCR amplification results.

To synthesize cDNA was using the ReverTra Ace (Toyobo, Osaka, Japan) according to manufacturer’s guidelines. Real-time qPCR was using the iTaq Universal SYBR Green Supermix (Bio Rad, Hercules, CA, USA) according to manufacturer’s guidelines and the solution was constructed as follows: 1 μL diluted cDNA was added to 5 μL iTaq Universal SYBR Green Supermix, 1 μL each of 5 pmol/μL diluted forward and reverse primers. C1000 Thermal Cycler (Bio Rad, Hercules, CA, USA) to measure the expression of target gene. Conditions used for real-time qPCR were as follows: incubation at 95°C for 5 min followed by 40 cycles of denaturation at 95°C for 10 s and annealing at 64°C for 30 s. All measurements were performed 3 times for each sample and relative gene expression was using the 2–ΔΔCt method [40 (link)]. Relative expression of the target gene was normalized to Glyceraldehyde 3-phosphate de12hydrogenase (GAPDH). The expression of DEGs was conducted by qRT-PCR using the primers listed in S7 Table.

The primers for the target and reference genes were designed using the online primer3 program (version 4.0.0)⁷ and are listed in Table 5. The total RNA from female adults was isolated and treated with RNase-free DNase I at 37°C for 30 min using the DNase I kit (Takara, Dalian, China). A reaction volume of 20 μl, which contained 200 nM of each forward and reverse primer, cDNA produced from 2 μg of total RNA, 8 μl of nuclease-free water, and 10 μl of 2X iTaq universal SYBR Green Supermix (BIO-RAD, CA, United States) was used. The real-time PCR analysis of 200 ng of total RNA was performed using a 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, United States). The qPCR reaction conditions were as follows: 95°C for 2 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The levels of the target genes were compared with those of the housekeeping gene β-actin using the 2^–ΔΔCt qPCR method. The means and standard errors for each time point were obtained from the average of four independent samples. The individual animals were randomized into two treatment groups, and three biological replications of each treatment were performed.

Total RNA from cultured cells was prepared using the TRIzol Reagent (Invitrogen/Thermo-Fisher) followed by a column clean-up using the PureLink RNA Mini Kit according to the manufacturer’s protocol for whole transcriptome isolation (Invitrogen). RNA concentration was quantitated using the NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies). Gene expression was measured by generating cDNA from 200 ng of total RNA using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA). The synthesized cDNA was diluted in 2X iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA) and combined with 10 μM of each forward and reverse primer. Specific primer sequences used are as follows: IGFBP4, forward 5′- AGCCCTCTGACAAGGACGAG-3′ and reverse 5′- TCCGGTCTCGAATTTTGGCG-3′; SLC2A1, forward 5’-CTGGCATCAACGCTGTCTTC-3′ and reverse 5′- GTTGACGATACCGGAGCCAA-3′; and the normalization control 36B4, forward 5′-ATCAACGGGTACAAACGAGTCCTG-3′ and reverse 5′- AAGGCAGATGGATCAGCCAAGAAG-3′. PCR reactions were run on the Bio-Rad CFX 96 Real-Time PCR (Bio-Rad, Hercules, CA) instrument under the following conditions: hold at 95 °C for 10 min, then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative gene expression was assessed using the differences in normalized Ct (ΔΔCt method) after normalization to 36B4.

Total RNA isolation, reverse transcription and the PCR primers were the same as described for RT-PCR. qPCR was run on a 96-well microtitration plate using a CFX96 TouchTM real-time PCR Detection System (Bio Rad, USA). PCR was run in a 10 μL solution containing 500 ng of cDNA, 5 μL of 2x iTaq Universal SYBR^® Green Supermix (Bio-Rad), 1 μL of primer solution at a concentration of 5 μmol/L and 2.5 μL of RNase-free UltraPureTM DEPC-treated water for 39 cycles at 57 °C. The samples were measured in triplicate.