Total RNA was extracted by Trizol (Beyotime, Beijing, China). According to the manufacturer’s protocols, a total of 500 ng of RNA was first reverse-transcribed into cDNA using BeyoRT™ II cDNA kit (Beyotime, Beijing, China). Next, qRT-PCR was performed using the BeyoFast™ SYBR Green qPCR Mix kit (Beyotime, Beijing, China). The relative expression of mRNA was calculated using 2(-ΔΔCt) method. The primers used are as follows: LEP, 5’-GCTGTGCCCATCCAAAAAGTCC-3’ (forward) and 5’-CCCAGGAATGAAGTCCAAACCG-3’ (reverse); NGF, 5’-ACCCGCAACATTACTGTGGACC-3’ (forward) and 5’-GACCTCGAAGTCCAGATCCTGA-3’ (reverse); PCOLCE2, 5’-GCAGTGAAGGTTTTCCTGGAGTG-3’ (forward) and 5’- AGTCATAGCGGCACAGGTTGTC-3’ (reverse). ACTB, 5’-CACCATTGGCAATGAGCGGTTC-3’ (forward) and 5’-AGGTCTTTGCGGATGTCCACGT-3’ (reverse).
Beyort 2 cdna kit
Manufactured by Beyotime
Sourced in China
The BeyoRT™ II cDNA kit is a laboratory product designed for the reverse transcription of RNA into cDNA. The kit includes reagents and protocols necessary for the conversion of RNA into complementary DNA (cDNA) molecules, which can then be used for various downstream applications such as gene expression analysis and sequencing.
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Lab products found in correlation
4 protocols using beyort 2 cdna kit
1
RNA Extraction and qRT-PCR Analysis
2
Quantitative Analysis of Gene Expression
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Total RNA from the stored plants was extracted with TRIzol, and cDNA was obtained using the BeyoRT™ II cDNA kit (Beyotime, Shanghai, China). Quantitative analysis was performed by real-time PCR in conjunction with Hieff® SYBR Green Master Mix (Yeasen) on a LightCycler 480 System (Roche, Vienna, Austria) according to the manufacturer’s instructions. The primers used are listed in Table S15 . The following reaction conditions were applied: predenaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and 65 °C for 60 s, and the melting curve was evaluated from 65 °C to 95 °C. The relative transcript levels of the candidate genes were calculated according to the 2−ΔΔCT method. The melting peaks of the candidate genes are displayed in Fig. S3 .
3
Quantitative Analysis of GmNTP Genes
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Total RNA from the stored plants was extracted with Trizol and cDNA was obtained using the BeyoRT™ II cDNA kit (Beyotime, Shanghai, China). The quantitative analysis was performed by real-time PCR in conjunction with Universal SYBR qPCR Mix (Biosharp, Hefei, Anhui, China) on a LightCycler 480 System (Roche, Vienna, Austria) in accordance with the manufacturer’s instructions. The primers used are listed in Table S1 . The following reaction conditions were applied: pre-degeneration at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 15 s and 60 °C for 30 s, and the melting curve was evaluated form 60 °C to 95 °C. The relative transcript levels of the GmNTP genes were calculated according to the 2−ΔΔCT method [83 (link)]. The melting peaks of the candidate genes are displayed in Figure S8 .
4
Quantitative Analysis of ATM Expression
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Total RNA was extracted from cells using the RNAeasy Kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions and was quantified on a OneDrop1000 UV spectrophotometer (Nanjing Zihanmu Scientific Instrument Co., Ltd.). The first strand of cDNA was synthesized according to the BeyoRT II cDNA kit (Beyotime Institute of Biotechnology) with 0.5-1.0 ng total RNA as the template. Subsequently, 1 µl cDNA underwent qPCR to detect target genes using the BeyoFast SYBR Green qPCR Mix (Beyotime Institute of Biotechnology) on the LineGene9600 (Hangzhou Bioer Co., Ltd.). The protocol for qPCR was as follows: 95°C for 5 min, followed by 95°C for 15 sec and 60°C for 20 sec for 40 cycles. GAPDH was used as the internal control. The relative expression of target genes was calculated by the 2−ΔΔCq method (15 (link)). The primers used were as follows: ATM, forward5′-TGGAAGCTGCTTGGGAGAAG-3′, reverse 5′-AGGCCAGCATTGGATCTGTT-3′; GAPDH, forward5′-GCACCGTCAAGGCTGAGAA-3′, reverse 5′-TAAGCAGTTGGTGGTGCAGG-3′.
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