Hiscript 3 rt supermix for qrt pcr

To analyze the effect of JH on the JH signal key gene in soldiers, Quantitative Reverse Transcription PCR was performed using the sample of the soldiers’ head on the 1st to 4th day after feeding JHA. Met, Kr-h1 and RaSsp1 were selected as JH key genes. The detail of qRT-PCR was the same as in a previous report [17 (link)]. RNA was extracted from 5 heads of soldiers with Trizol RNA extraction reagent (Thermo, Carlsbad, CA, USA), and then cDNA was obtained using the reverse transcription kit HiScript III RT SuperMix for qRT-PCR (Vazyme Bio, Nanjing, China). qRT-PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR system (ABI, Foster City, CA, USA). Ribosomal protein gene L13a (RPL13a) and elongation factor 1α (EF1-α) mRNA levels were used as references. The specific primers were as follows, RaSsp1: F 5′-ACTGTGCTTGGCGCTGTC-3′, R 5′-CTGGGATGTGGTATTGCTT T-3′; Met: F 5′-GCCCTCATCATCCGCCTT, R 5′-CTTGCCATCACGAGAACG-3′; Kr-h1: F 5′-AGCAGCCCAGATTTACCT, R 5′-GTCTTCGCCCTCCTTTCC-3′; RPL13a: F 5′-TCTGTGGAGGACGGTTAG, R 5′-ACTTTCTGCCTGGTTTCA-3′; and EF1-alfa: F 5′-CCCTTCGTCTTCCTCTTC, R 5′-CTCCAGCGACATAACCAG-3′. Each experiment was performed with three biological replicates, and three qRT-PCR analyses were performed using the same cDNA sample of each time point.

Total RNA was extracted from cells in the CYP27A1 WT and KO groups (n = 3 per group) using Trizol reagent. The concentration and purity of total RNA were detected using a Nanodrop 2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA). Reverse transcription was preformed to synthesize cDNA using Hiscript®iii Rt Supermix for qRT-PCR (RC323-01; Nanjing Vazyme Biotech Co, Nanjing, China). The cDNA was used as the template to amplify targeted sequences with specific primers using the Chamq Universal SYBR qRT-PCR Master Mix (Q711-02; Nanjing Vazyme Biotech Co, Nanjing, China). The results were analyzed by the 2^−ΔΔCT method (Livak & Schmittgen, 2001 (link)). The sequences of the eight primers were shown in Table 1.

Total RNA was extracted using TRizol Reagent (Sigma-Aldrich Co. LLC.) according to the manufacturer’s instructions. HiScript III RT SuperMix for qRT-PCR (Vazyme Biotech Co.) was used to synthesize complementary DNA from 1 μg of the RNA sample. β-actin gene was used as the internal control. qRT-PCR was performed using SYBR Green mix (Vazyme Biotech Co.) to evaluate gene expression levels. All qRT-PCR experiments were performed in duplicate. Relative gene expression levels were calculated using the standard 2−ΔΔCt method, followed by statistical analysis using GraphPad Prism 9.0 software. Mann–Whitney U test was used for comparison between the two groups as the data did not conform to normal distribution. Primers used in this study are listed in Table 3. qRT-PCR results have been uploaded to Supplementary Table 1.