Cfx96 real time pcr detection system thermal cycler

qRT-PCR was performed to validate the accuracy of the LFQ analysis. Total RNA was extracted from a randomly selected pupa in each group using TRIZOL Reagent (Life technologies, Carlsbad, CA, US). The first-strand cDNA was synthesized using PrimeScript^™ RT Master Mix (Perfect Real Time) Kit (Takara, Shiga, Japan). Sixty-four pairs of specific primers were designed to amplify the randomly selected genes encoding DAPs determined by LFQ data (S1 Table), and UBQ was used as a reference gene [41 ]. qRT-PCR was performed with a CFX96^™ Real-Time PCR Detection System thermal cycler (Bio-Rad, Hercules, CA, USA) in a reaction volume of 10 μL, containing 5 μL SYBR^® Premix Ex Taq II (Takara), 0.5 μL forward and reverse primers (10 μM), 1μL cDNA, and 3 μL ddH₂O. The reaction conditions were as follows: 95°C for 30s; followed by 40 cycles of 95°C for 5s and 60°C for 30s. A relative standard curve was constructed based on a series of 10-fold diluted cDNA templates to estimate the amplification efficiency (E = 10^−1/slope) of each pair of primers (S1 Table). Pearson’s r correlation coefficient was calculated to evaluate the correlation between the qRT-PCR and LFQ data. Three biological and three technical replicates were set for each gene.

qRT-PCR was performed to verify the accuracy of the DEG analysis. Total RNA from the five pupal stages described above was extracted using TRIZOL Reagent. The first-strand cDNA was synthesized using PrimeScript™ RT Master Mix (Perfect Real Time) Kit (Takara, Shiga, Japan). Twenty-one pairs of specific primers were designed to amplify the genes selected from multiple comparison (S1 Table). Ubiquitin was used as a reference gene for normalization [40 ]. qRT-PCR was conducted in 25 μL volumes containing 12.5 μL SYBR^® Premix Ex Taq II (Takara), 2 μL primers (10 μM), 1μL cDNA, and 9.5 μL ddH₂O, using a CFX96™ Real-Time PCR Detection System thermal cycler (BIO-RAD, Hercules, CA, USA). Amplification conditions were as follows: initial denaturation at 95°C for 30s; followed by 40 cycles of denaturation at 95°C for 5s, 60°C for 30s. Pearson’s r correlation coefficient was calculated to evaluate the correlation between the qRT-PCR and DEG data. Three biological and three technical replicates were performed for each gene.

After the isolation of the genetic material from whole blood, we proceeded to genotype all the samples for all chosen genetic variants according to the protocol described in previous our publications [27 (link), 28 (link)]. Briefly, real-time PCR was performed in a CFX96™ Real-Time PCR Detection System Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA), using the TaqMan allele-specific discrimination assay, TaqMan® SNP Genotyping Assay (Thermo Fisher Scientific, Waltham, MA, USA), and RT PCR Mix Probe (A&A Biotechnology, Gdynia, Poland). Moreover, the CFX Manager TM Software (version 3.1) was used to analyse the obtained results. TaqMan® SNP Genotyping Assay details are included in Table 2.
A representative allelic discrimination X–Y scatter-plot of the c.+396 T>G SNP (rs2227307) of the IL-8 is presented in Fig 1.