TRIzol reagent was used to isolate the total RNA of SMGs tissues according to the manufacturer's protocol. The RevertAid First Strand cDNA Synthesis Kit (Promega, Madison, WI, USA) was used to synthesize cDNA from 2 μg of total RNA. qRT-PCR was carried out by using FastStart universal SYBR Green Master (ROX) on a BioRad CFX96 system (Thermo Fisher Scientific). β-Actin served as an endogenous control. The sequences of the primers are listed in Supplementary Tables S1 and S2 .
Biorad cfx96 system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BioRad CFX96 system is a real-time PCR detection system used for gene expression analysis, genotyping, and other molecular biology applications. It features a 96-well plate format and can perform thermal cycling and fluorescence detection.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Sybr green master mix, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Revertaid first strand cdna synthesis kit, by Promega (1 mentions)
Sybr green real time pcr kit, by Thermo Fisher Scientific (1 mentions)
Cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using biorad cfx96 system
1
Isolation and Quantification of SMG RNA
2
Quantitative RT-PCR Analysis of ACT2 Gene
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Total RNA from tissues was extracted with TRIzol Reagent (Thermo Fisher Scientific), 2 µg total RNA were treated with DNAse I (Thermo Fisher Scientific), and reverse transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions (Thermo Fisher Scientific). The ACT2 gene was used as an internal control. qRT-PCR analysis was performed on a Bio-Rad CFX96 system with SYBR Green Master Mix (Thermo Fisher Scientific). Primers are listed in Table S2. All experiments were repeated at least three times each in technical triplicates.
3
Quantitative RNA Expression Analysis
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TRIzol reagent from Invitrogen was leveraged for the extraction of RNA, which was then reversely transcribed into complementary DNA (cDNA) with cDNA Reverse Transcription Kit (Invitrogen). Then, quantitative real-time polymerase chain reaction (qRT-PCR) was conducted with SYBR Green Real-Time PCR Kit (Thermo Fisher Scientific, Waltham, MA, USA) on the Bio-Rad CFX96 System (Thermo Fisher Scientific). The 2−ΔΔCT method was selected for transcript quantification. As the internal control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/U1 was adopted.
4
Plant RNA Extraction and RT-qPCR Analysis
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RNA extraction and RT-qPCR analysis were performed with Zuo et al. method with a few modifications73 . Total RNA from plant tissues were extracted with TRIzol Reagent (Invitrogen, ThermoFisher, USA), 1 μg total RNA was treated with DNAse I (Thermo Scientific) and reverse transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions (Thermo Scientific). The constitutively expressed gene ACT2 encoding the actin monomer was used as an internal control. qPCR was performed on a Bio-RAD CFX96 system with SYBR Green master mix (Thermo Scientific). Primers are listed in Supplementary Table 1 .
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