Maize stalk tissues were collected and frozen immediately in liquid nitrogen, and total RNA samples were isolated using the Plant RNA extraction kit (TIANDZ, Inc., Beijing, China). One microgram of total RNA was converted to cDNA using HiScript II RT SuperMix for qPCR (Vazyme, Nanjing, China) according to the manufacturer’s instructions. qRT-PCR reactions were performed with the Bio-Rad CFX96™ real-time PCR system using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). Maize Ubc9 was used as an internal control (forward primer, 5′-acgaaggtcttccatccaaacatc-3′; reverse primer, 5′-gtttcatgggcaacaccacaatcg-3′), and the relative expression levels for target genes were calculated using the 2–ΔΔCt method. qRT-PCRs were all performed with three biological replicates. The primers used for ZmVPS37A are as follows: ZmVPS37A-F 5′-gagctggagatttcctctcttc-3′ and ZmVPS37A-R 5′-gaagactgcggactatctgaagc-3′.
Cfx96 real time pcr system
Manufactured by Vazyme
Sourced in China
The CFX96™ real-time PCR system is a thermal cycler designed for real-time PCR analysis. It is capable of performing precise temperature control and fluorescence detection for DNA amplification and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Chamq sybr qpcr master mix, by Vazyme (2 mentions)
Hiscript 2 rt supermix for qpcr, by Vazyme (1 mentions)
Plant rna extraction kit, by Tiandz (1 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (1 mentions)
Hifair 3 1st strand cdna synthesis kit, by Yeasen (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Hiscript 2 q select rt supermix for qpcr gdna wiper kit, by Vazyme (1 mentions)
T5 fast qpcr mix sybrgreeni kit, by Vazyme (1 mentions)
Trizol reagent, by Vazyme (1 mentions)
Fastpure plant total rna isolation kit, by Vazyme (1 mentions)
Hiscript 3 rt supermix for qpcr gdna wiper, by Vazyme (1 mentions)
Nanodrop system, by Thermo Fisher Scientific (1 mentions)
Rnaiso plus kit, by Takara Bio (1 mentions)
Chamq universal sybr qpcr master mix kit, by Vazyme (1 mentions)
5 protocols using cfx96 real time pcr system
1
Maize Transcriptional Analysis via qRT-PCR
2
RNA extraction and qRT-PCR analysis
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According to the manufacturer’s instructions, total RNA was extracted from all samples using the RNAprep pure Plant Kit (TIANGEN, Beijing, China). For qRT-PCR analysis, first-strand cDNAs were synthesized from DNaseI-treated total RNA using the Hifair® III 1st Strand cDNA Synthesis kit (Yeasen, Shanghai, China) according to the manufacturer’s instructions. qRT-PCR was performed on the Biorad CFX96 real-time PCR system using the ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China). The relative expression levels were calculated by using 2−∆∆Ct method. The qRT-PCR assays were performed with three biological and technical replicates. The gene-specific primers are listed in Supplementary Additional file 1: Table S2 .
3
Gene Expression Analysis by qRT-PCR
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Total RNA was extracted by using the TRIzol reagent (Vazyme Biotech Co. Ltd., Nanjing, China). DNaseI-treated total RNAs were subjected to reverse transcription (RT) with the HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotech Co. Ltd., Nanjing, China). Transcript levels of selected genes were measured by qRT-PCR, using the CFX96® Real-Time PCR System using the 2 × T5 Fast qPCR Mix (SYBRGreenI) kit (Vazyme Biotech Co. Ltd., Nanjing, China). Rice actin gene (Rac1) was used for normalization. The ΔΔCt method was used to calculate the relative transcript abundance [78 (link)]. The primers for qRT-PCR are given in Table S1 .
4
Cloning and Expression Analysis of CscpFtsY
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Total RNA was extracted from the root, stem, leaf, male flower (anthesis), ovary (one day before anthesis), cotyledon of the CCMC and yl plant by FastPure Plant Total RNA Isolation Kit (Polysaccharides & Polyphenolics – rich) (Vazyme) for cloning the gene and analyzing the relative expression level of CscpFtsY. The first strand cDNA was synthesized by HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme). The cDNA from the leaf of CCMC and yl plants was used as a template for cloning the target gene. Primers used for amplification of the full-length cDNA of the CscpFtsY gene are listed in Table S2 .
qRT-PCR was performed in a 96-well plate using a Bio-Rad CFX 96 Real-Time PCR System with ChamQ SYBR qPCR Master Mix (Vazyme). The cucumber CsaACTIN2 was used as the reference gene to normalize the gene expression results. The amplification was initiated by heating to 94 °C for 10 min, followed by 40 cycles of 94 °C for 5 s and 57 °C for 30 s. All experiments were performed with three biological replicates and three technical replicates. Gene expression level at different organs was calculated by the basis of the 2–ΔΔCt and the values represented the n-fold difference of yl relative to the gene expression of WT. The primers used are listed in Table S2 .
qRT-PCR was performed in a 96-well plate using a Bio-Rad CFX 96 Real-Time PCR System with ChamQ SYBR qPCR Master Mix (Vazyme). The cucumber CsaACTIN2 was used as the reference gene to normalize the gene expression results. The amplification was initiated by heating to 94 °C for 10 min, followed by 40 cycles of 94 °C for 5 s and 57 °C for 30 s. All experiments were performed with three biological replicates and three technical replicates. Gene expression level at different organs was calculated by the basis of the 2–ΔΔCt and the values represented the n-fold difference of yl relative to the gene expression of WT. The primers used are listed in Table S
5
Quantification of mRNA Expression in Tissues
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Total mRNAs in the intestine and liver tissues were extracted using RNAiso plus kit (TaKaRa, China), and then, the mRNA concentration was measured using a Nanodrop system (DN-2000; Thermo Scientific). cDNA was synthesized using HiScript III RT SuperMix cDNA synthesis kits (Vazyme Biotech, Nanjing, China). Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR; total volume = 20 µl) was performed on a Bio-Rad CFX96 Real-Time PCR System using ChamQ Universal SYBR qPCR Master Mix kit (Vazyme Biotech, Nanjing, China). The program for qPCR herein was as follows: 95°C for 30 s (pre-denaturation); 95°C for 5 s (denaturation), followed by 60°C for 30 s (40 cycles); finally, 95°C for 10 s. RT-qPCR–specific primers used here have been designed in previous studies (Xu et al., 2016 (link); Wei et al., 2020 (link)) and synthesized by Shanghai Biological Engineering (Table 2 ). The relative mRNA expression was determined using the 2−ΔΔCt method.
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