After the stress of 4 h and 58 h, total RNA was extracted from pepper leaves with an RNA concentration of 1.0 (Tianenze, Beijing, China). Each group has three biological replicates. Primers were designed according to Premier 6.0 (S5). cDNA was synthesized using the reverse transcriptase MMLV kit (China ABM). Real-time quantification was performed using the CFX Manager (Bio-Rad, USA) and the SYBR Green Real-Time PCR Master Mix (Abm, Canada). The protocol of real-time PCR was as follows: predenaturation at 95°C for 10 minutes, then 40 rounds of amplification at 40°C, 15 s denaturation at 95°C, and annealed at 30°C for 30 s, extended to 72°C and read the plate to record fluorescence data at 65°C. Melting curves were performed at 65°C to 95°C to check the specificity for the amplified products. Each reaction was repeated three times. Pepper ACTIN1 was used as an internal control.
Sybr green real time pcr master mix
Manufactured by Applied Biological Materials
Sourced in Canada
SYBR Green Real-Time PCR Master Mix is a ready-to-use solution for quantitative real-time PCR analysis. It contains SYBR Green I, a fluorescent dye that binds to double-stranded DNA, enabling real-time detection and quantification of PCR products.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Hiscript 3 1st strand cdna synthesis kit, by Vazyme (2 mentions)
Cfx manager, by Bio-Rad (1 mentions)
Trizol reagent, by Tiangen Biotech (1 mentions)
Reverse transcription kit, by Promega (1 mentions)
Cfx96 real time pcr system, by Bio-Rad (1 mentions)
Abi quantstudio 3 real time pcr system, by Thermo Fisher Scientific (1 mentions)
5 protocols using sybr green real time pcr master mix
1
Pepper Leaf RNA Extraction and qPCR Analysis
2
Quantitative PCR Protocol for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For qPCR assays, reverse transcription total RNA was isolated using TRIzol reagent (#W9514, Tiangen) from either tissue samples or cultured cells. Total RNA was obtained and then reverse-transcribed was performed with 2 μg total RNA using the Reverse Transcription Kit (#PIA279, Promega) following the manufacturer’s instructions. qRT-PCR experiments were carried out by an CFX96 real-time PCR system (Bio-Rad, C1000) using SYBR green real-time PCR master mix (G891, abm). The mRNA expression levels of the target genes were normalized to Gapdh or actin expression. The primer pairs used in this study are listed in Table 1 .
3
TRIZOL RNA Extraction and qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. And cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (G592, abm, Canada) following the manufacturer’s protocol. The mRNA levels were detected by SYBR Green real-time PCR Master Mix (G891, abm, Canada) and performed on ABI QuantStudio 3 Real-Time PCR System (Applied Biosystems). The mRNA expression was normalized by the expression of GAPDH and relative expression levels were calculated using the 2^-ΔΔCT method in cell and tissue lysates. Primers are shown in Supplementary Table S3 .
4
Quantitative Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the targeted cells using TRIzol reagent (Invitrogen) in accordance with the manufacturer's instructions. One Microgram total RNA was reverse transcribed to cDNA using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme). The relative mRNA level of targeted gene was measured with qPCR using SYBR green real-time PCR master mix (Applied Biological Materials Inc) with specific primer (Supplementary Table 1 ). All reactions were performed in triplicate. The mRNA level of housekeeping gene GAPDH was used as an internal control. Relative gene fold was determined with the comparative cycle threshold (2−ΔΔCT) method.
5
Real-Time qPCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the targeted cells using TRIzol reagent (Invitrogen) in accordance with the manufacturer’s instructions. 1 µg total RNA was reverse transcribed to cDNA using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme). The relative mRNA level of targeted genes was measured with qPCR using SYBR Green real-time PCR master mix (Applied Biological Materials Inc) with specific prime. All reactions were performed in triplicate. The mRNA level of housekeeping gene GAPDH was used as an internal control. The relative gene fold was determined with the comparative cycle threshold (2-ΔΔCT) method.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!