One microgram of the total RNA was subjected to reverse transcription using All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech Co., Ltd. Beijing, China). Transcription levels of silicon-related genes and genes related to increasing rice resistance under arsenic stress were determined by quantitative RT-PCR. The sequence of primers used in this experiment is given in Table 1 , and Actin-1(Os03g0718100) was used as the reference gene. Primers were designed for qRT-PCR analysis by online tools on the https://biodb.swu.edu.cn/qprimerdb/ (Accessed on 20 January 2020). The qRT-PCR reaction system was prepared by TransStart Tip Green qPCR SuperMix and an Eppendorf realplex4 instrument. The reaction process was as follows: initial denaturation at 94 °C for 30 s, denaturation at 94 °C for 5 s, annealing at 53 °C for 15 s, and extension at 72 °C for 10 s. When amplification was completed, product characteristics were determined based on the melting curve. Each candidate gene was performed with four independent reactions. The relative expression of the gene was calculated by the 2−∆∆Ct method and by the threshold cycle values (Ct) of each candidate gene in both CK and experimental samples [28 (link)].
Tip green qpcr supermix
Manufactured by Eppendorf
Tip Green qPCR SuperMix is a ready-to-use master mix for quantitative real-time PCR (qPCR) applications. It contains all the necessary components, including a DNA polymerase, buffer, dNTPs, and a green fluorescent dye for signal detection.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using tip green qpcr supermix
1
Quantitative Analysis of Silicon-Related Genes under Arsenic Stress in Rice
2
Transcriptional Response of Rice to Cold Stress
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Total RNA from Dular and Lsi1-OX rice exposed to a temperature of 15 °C for 12, 24 and 36 h was extracted using Trizol and reverse-transcribed into cDNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix. The control groups were grown at 26 °C for 12, 24 and 36 h. Specific primers for tubulin/FtsZ domain containing protein (LOC_Os03g51600, LOC_Os05g34170, LOC_Os07g38730), histone H1 and nucleic acid binding protein (NABP) are listed in Table S3 ; the β-actin gene was taken as the reference. The qPCR reaction system was prepared using TransStart Tip Green qPCR SuperMix and an Eppendorf realplex4 instrument. The reaction process was as follows: pre-denaturation at 94 °C for 30 s, denaturation at 94 °C for 5 s, annealing at 55 °C for 15 s, extension at 72 °C for 10 s; 42 cycles. When the amplification was finished, analysing of the melting curve was conducted and specificity of the product was determined based on the melting curve. Each candidate mRNA was set with four independent replicates. The relative expression of the gene was calculated by the 2-△△Ct method with the threshold cycle values (Ct) of each candidate mRNA in both the control and test samples [37 (link)].
3
Transcriptional Response of Rice to Cold Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from Dular and Lsi1-OX rice exposed to a temperature of 15 °C for 12, 24 and 36 h was extracted using Trizol and reverse-transcribed into cDNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix. The control groups were grown at 26 °C for 12, 24 and 36 h. Speci c primers for tubulin/FtsZ domain containing protein (LOC_Os03g51600, LOC_Os05g34170, LOC_Os07g38730), histone H1 and nucleic acid binding protein (NABP) are listed in Table S3; the β-actin gene was taken as the reference. The qPCR reaction system was prepared using TransStart Tip Green qPCR SuperMix and an Eppendorf realplex 4 instrument. The reaction process was as follows: pre-denaturation at 94 °C for 30 s, denaturation at 94 °C for 5 s, annealing at 55 °C for 15 s, extension at 72 °C for 10 s; 42 cycles. After ampli cation, a melting curve analysis was performed to verify the speci city of the ampli ed products, under the following conditions: 95 °C for 15 s, 60 °C for 15 s and an increase to 95 °C over a 10-min period, followed by a 15-s holding period. Threshold cycle values (Ct) were recorded for each candidate mRNA in both the control and test samples. Each candidate mRNA was set with four independent replicates. The relative expression of the gene was calculated by the 2 -△△Ct method [40] .
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