Total RNA was extracted from mud crab hemocytes by using TRIzol (Cwbio, Beijing, China) according to the manufacturer’s protocol, followed by cDNA synthesis with the QuantScript RT kit (Tiangen, China), and the cDNA was used as a template for qPCR using the SYBR green system (Tiangen, China). Gene-specific primers are listed as follows: HUWE1-F (5′-GGCTTATCTACCTGATGGAACACA -3′), HUWE1-R (5′-AGGAATATTGCGTCCGTTGG -3′), TRAF6-F (5′-CCAATTGACAA CACCCCTCTG -3′), TRAF6-R (5′-GGCGGAACACTCATTCGGAC -3′), p53-F (5′-CAGGAGGTGCTAATAAGGGTAACG -3′), and p53-R (5′-TTCACAACGATGGGAGGG GT-3′). The primers β-actin-F (5′-GCGGCAGTGGTCATCTCCT -3′) and β-actin-R (5′-GCCCTTCCTCACGCTATCCT -3′) were used to quantify the internal control (β-actin). Relative fold change was analyzed by the threshold cycle (2−ΔΔCT) algorithm (53 (link)).
Sybr green system
Manufactured by Tiangen Biotech
Sourced in China, United States
The SYBR green system is a real-time PCR detection system that utilizes SYBR green dye to monitor the amplification of DNA during the PCR process. The SYBR green dye binds to double-stranded DNA, resulting in an increase in fluorescence intensity that is proportional to the amount of DNA present. This system provides a simple and cost-effective method for quantifying gene expression or detecting the presence of specific DNA sequences.
Automatically generated - may contain errors
2 protocols using sybr green system
1
Quantitative PCR Analysis of Mud Crab Immunity
2
Extraction and Expression Analysis of PmSPX Genes
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Total RNA was isolated from plant tissues using RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer's protocols. RNA concentration and purity were measured with IMPLEN GMBH (NanoPhotometer N60 Touch, Germany), and then the integrity of RNA was identified by gel electrophoresis. The first-strand cDNA was synthesized using the FastKing gDNA Dispelling RT SuperMix (Trangen, Beijing, China). The specific primers used in this study were designed based on the P. massoniana SPX genes sequences using Primer Primer 5.0 software and synthesized by Sangon Biotech Company (Shanghai, China) (Table S3). RT-qPCR was executed on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the SYBR Green system (Tiangen, Beijing, China). The P. massoniana UBC gene was employed as a control. In this study, three independent biological replicates and three technical replicates for each biological replicate were examined. The relative expression levels of PmSPX genes were calculated by the 2 -∆∆CT method [41] (link).
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