qPCR was conducted to validate the expression levels of 25 randomly selected potential regulatory DEGs. One microgram of RNA, treated with gDNA-remover to eliminate genomic DNA, was reverse transcribed using an oligo(dT) primer according to the manufacturer’s protocol (HiScript III 1st Strand cDNA Synthesis Kit; Vazyme, China). qPCR analysis was performed with the ChamQ SYBR Green master mix (Vazyme, China) using LightCycler 96 (Jena, Germany) under the following cycling conditions: pre-denaturation at 95 °C for 30 s, followed 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Each treatment group had three biological replicates, and the experiment was performed in triplicate. Melting curve analysis was performed, and the absence of non-specific products and primer dimers was verified. Relative expression levels were calculated using the 2–△△Ct method. Expression levels of the target genes were normalised to those of the internal control gene, Actin [27 (link)]. The genes and primers used are shown in S3 Table .
Chamq sybr green master mix
Manufactured by Vazyme
Sourced in China, Switzerland
ChamQ SYBR Green master mix is a ready-to-use solution designed for real-time quantitative PCR (qPCR) and reverse transcription-quantitative PCR (RT-qPCR) applications. It contains SYBR Green I, a fluorescent dye that binds to double-stranded DNA, enabling the detection and quantification of target DNA sequences.
Automatically generated - may contain errors
4 protocols using chamq sybr green master mix
1
Validating Gene Expression Profiles by qPCR
2
Validation of Differentially Expressed Genes by qRT-PCR
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Twenty-one genes, including 15 DEGs, were randomly selected, and 6 Pb-regulated genes were used for further validation by qRT-PCR. Total RNA extraction and genomic DNA removal were performed as described above. First-strand cDNA was synthesized from 1 μg of RNA using a HiScript® 1st Strand cDNA Synthesis kit (Vazyme, China). The cDNA products were then diluted tenfold with nuclease-free water for use as a template for qRT-PCR, which was performed using the ChamQ™ SYBR Green Master mix (Vazyme, China) with a CFX96 Real-Time PCR Detection System (Bio-Rad). The specific primer pairs used for qRT-PCR are listed in Additional file 8 : Table S5. For qRT-PCR validation, the primer specificity was tested by PCR (Additional file 9 : Figure S4). All samples were normalized to the CACS gene [73 (link)]. The DEG expression fold change was calculated based on the threshold cycle (Ct), where ΔCt = Cttarget − CtCACS and Δ (ΔCt) = ΔCtControl − ΔCtIndicated condition.
3
Validating Transcriptomic Data by qRT-PCR
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To further validate the veracity and reliability of transcriptomic data, quantitative real-time PCR was preformed, and phosphoserine aminotransferase 1 (psat1), serine hydroxymethyltransferase (shmt), methionine synthase (mtr), tropomyosin-1 (tpm1) and phosphate cytidylyltransferase 1 (pcyt1) were selected for qRT-PCR analysis. The housekeeping gene β-actin was used as an internal normalization control, and the specific primers for the candidate genes used for qPCR were designed by Primer Premier 5.0 (Table S3). The qPCR was carried out in a quantitative thermal cycler system (Light cycler® 96, Roche, Switzerland) with each 20 µl reaction volume containing 10 μl of 2 × ChamQ SYBR Green Master Mix (Vazyme), 0.4 μl (each) gene-specific forward and reverse primers (10 μm), 8.4 μl DEPC water and 0.8 μl of 1:4 diluted cDNA. The quantitative PCR program was 95 °C for 2 min, followed by 45 cycles of 95 °C for 10 s, 58 °C for 10 s and 72 °C for 20 s. The amplification efficiency was measured as following: E=10 (-1/slope) -1, and amplification efficiencies of all genes ranged from 98.4 % to 105.7 %.
The relative gene expression values were normalized by β-actin-expressed transcripts, and calculated using the 2 -∆∆Ct method as described by Livak and Schmittgen (2001) (link).
The relative gene expression values were normalized by β-actin-expressed transcripts, and calculated using the 2 -∆∆Ct method as described by Livak and Schmittgen (2001) (link).
4
Validating Transcriptomic Data by qRT-PCR
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To further validate the veracity and reliability of transcriptomic data, quantitative real-time PCR was preformed, and phosphoserine aminotransferase 1 (psat1), serine hydroxymethyltransferase (shmt), methionine synthase (mtr), tropomyosin-1 (tpm1) and phosphate cytidylyltransferase 1 (pcyt1) were selected for qRT-PCR analysis. The housekeeping gene β-actin was used as an internal normalization control, and the specific primers for the candidate genes used for qPCR were designed by Primer Premier 5.0 (Table S3). The qPCR was carried out in a quantitative thermal cycler system (Light cycler® 96, Roche, Switzerland) with each 20 µl reaction volume containing 10 μl of 2 × ChamQ SYBR Green Master Mix (Vazyme), 0.4 μl (each) gene-specific forward and reverse primers (10 μm), 8.4 μl DEPC water and 0.8 μl of 1:4 diluted cDNA. The quantitative PCR program was 95 °C for 2 min, followed by 45 cycles of 95 °C for 10 s, 58 °C for 10 s and 72 °C for 20 s. The amplification efficiency was measured as following: E=10 (-1/slope) -1, and amplification efficiencies of all genes ranged from 98.4 % to 105.7 %.
The relative gene expression values were normalized by β-actin-expressed transcripts, and calculated using the 2 -∆∆Ct method as described by Livak and Schmittgen (2001) (link).
The relative gene expression values were normalized by β-actin-expressed transcripts, and calculated using the 2 -∆∆Ct method as described by Livak and Schmittgen (2001) (link).
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