Nine candidate genes were selected for qRT-PCR validation. After extracting total RNA from breast muscle and leg muscle tissue, the total RNA reverse transcriptase kit (Takara, Kusatsu, China) was used to synthesize cDNA. Real-time PCR ABI 7500 Fast Real-Time PCR System using SYBR premix Ex TaqTM II (Takara, Kusatsu, China) was used to perform real-time PCR. The 2−ΔΔCT method was used to determine relative expression, and β-actin was used as the internal control for normalization of the results. All primer sequences were listed shown in Table S3 of the Supplementary Materials .
Total rna reverse transcriptase kit
Manufactured by Takara Bio
Sourced in China
The Total RNA reverse transcriptase kit is a laboratory tool designed to convert total RNA into complementary DNA (cDNA) through the process of reverse transcription. This kit provides the necessary reagents and enzymes to perform this essential step in various molecular biology applications, such as gene expression analysis, cDNA library construction, and RT-PCR.
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4 protocols using total rna reverse transcriptase kit
1
qRT-PCR Validation of Candidate Genes
2
Bone Metabolism Gene Expression Analysis
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Seven bone metabolism-related genes (Table S1 ) were selected for qRT-PCR. Total RNA was extracted from thoracic vertebrae and leg cartilage tissues. The total RNA reverse transcriptase kit (Takara, China) was used to synthesize cDNA. Real-time PCR ABI 7500 Fast Real-Time PCR System using SYBR premix Ex TaqTM II (Takara, Treasure Bioengineering Co., Ltd, Dalian, China) was used to perform real-time PCR. The 2−ΔΔCT method was used to determine relative expression, and β-actin was used as the internal control for the normalization of the results.
3
Validating Gene Expression in Chicken Muscles
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Seven candidate genes were selected for qRT-PCR validation. After extracting total RNA from breast muscle and leg muscle tissue, the total RNA reverse transcriptase kit (Takara, Kusatsu, Japan) was used to synthesize cDNA. Real-time PCR ABI 7500 Fast Real-Time PCR System using SYBR premix Ex TaqTM II (Takara, Kusatsu, Japan) was used to perform real-time PCR. The 2−∆∆CT method was used to determine relative expression, and ACTB was used as the internal control for normalization of the results. The experiment was repeated in triplicate. All primer sequences were listed shown in Table 3 .
4
Gene Expression Analysis by qRT-PCR
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The expression of CSRP3, KY, FHL1, LMCD1, and MYOZ2 genes was measured by qRT-PCR to verify the accuracy of transcriptome sequencing data by RNA-Seq. After extracting total RNA from hepatic tissue samples, cDNA was synthetized using the total RNA reverse transcriptase kit (Takara, Dalian, China). Real-time PCR was performed on an ABI 7500 Fast Real-Time PCR system using SYBR premix Ex Taq™ Ⅱ (Takara). The optimized cycling conditions were as follows: denaturation at 94°C for 5 min followed by 45 cycles of 94°C for 15 s and 55°C for 15 s. Each sample was tested in triplicate. The relative expression was determined using the 2−ΔΔCt method (Schmittgen and Livak, 2008 (link)), and β-actin was used as the internal control for normalization of the results. The sequences of primers for the genes tested were specifically designed according to the sequences located in GenBank (Table 2 ).
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