Total RNA was extracted from the shrimp muscle using total RNA isolation kit (Sangon Biotech (Shanghai) Co., Ltd., China). The integrity and concentration of RNA were detected by 1% agarose gel electrophoresis and GeneQuant pro (GE Pharmacia, USA), respectively. The isolated RNA was then reversely transcribed to cDNA by reverse transcription kit (Vazyme Biotech Co., Ltd., China).
The amplification reactions for quantitative real-time polymerase chain reaction (qRT-PCR) were performed in 96-well plates using a total volume of 20 μL, containing 0.4 μL of each primer, 4 μL of cDNA template, 10 μL of 2 × ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., China), 0.4 μL of ROX Reference Dye (Vazyme Biotech Co., Ltd., China), and 4.8 μL of sterilized double-distilled water. The qRT-PCR was performed with an ABI StepOnePlus Real-Time PCR System using the following cycle conditions: 95°C for 30 s, followed by 40 cycles of 95°C for10 s, and 60°C for 30 s [34 (link)]. Melting curve analysis was carried out to validate that only one PCR product was obtained in these reactions. The expression levels of genes were quantified relative to the expression of β-actin using the comparative CT method (2−△△CT method) [35 (link)]. The expression of β-actin was stable among the treatments. The gene expression was normalized with the LP group as control. The primer sequences of target genes (target of rapamycin (tor), ribosomal protein S6 kinase (s6k), eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4e-bp1), phosphatidylinositol 3-kinase (pi3k),and serine/threonine-protein kinase (akt)) were designed based on our transcriptome unigenes of kuruma shrimp (Table 3 ).
The amplification reactions for quantitative real-time polymerase chain reaction (qRT-PCR) were performed in 96-well plates using a total volume of 20 μL, containing 0.4 μL of each primer, 4 μL of cDNA template, 10 μL of 2 × ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., China), 0.4 μL of ROX Reference Dye (Vazyme Biotech Co., Ltd., China), and 4.8 μL of sterilized double-distilled water. The qRT-PCR was performed with an ABI StepOnePlus Real-Time PCR System using the following cycle conditions: 95°C for 30 s, followed by 40 cycles of 95°C for10 s, and 60°C for 30 s [34 (link)]. Melting curve analysis was carried out to validate that only one PCR product was obtained in these reactions. The expression levels of genes were quantified relative to the expression of β-actin using the comparative CT method (2−△△CT method) [35 (link)]. The expression of β-actin was stable among the treatments. The gene expression was normalized with the LP group as control. The primer sequences of target genes (target of rapamycin (tor), ribosomal protein S6 kinase (s6k), eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4e-bp1), phosphatidylinositol 3-kinase (pi3k),and serine/threonine-protein kinase (akt)) were designed based on our transcriptome unigenes of kuruma shrimp (
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