Total RNA was extracted from cells at 90% confluence by TRIzol (Invitrogen Life Technologies, Carlsbad, USA). Total RNA concentrations were measured using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, USA). According to the protocol, 2 μg of total RNA in each sample was reverse-transcribed by using the Reverse Transcriptase M-MLV (RNase H) Kit (Takara Biotechnology, Otsu, Japan). The cDNA after reverse transcription was diluted 5 times with ddH2O and stored at −20°C. qRT-PCR was performed using a SYBR Green RT-PCR Kit (Takara Biotechnology, Otsu, Japan), and specific primers were run in the CFX Connect™ Real-Time System. The relative expression of target genes was normalized to those of GAPDH and expressed as 2−ΔΔCt. The sequences of primers for qRT-PCR are listed in Table 1 .
Cfx connect real time system
Manufactured by Takara Bio
Sourced in Japan
The CFX Connect Real-Time System is a compact and versatile real-time PCR detection system. It is designed for various applications in molecular biology research, including gene expression analysis, genotyping, and pathogen detection. The system features a thermal cycler with a 96-well sample block and a sensitive optical detection system to monitor fluorescence signals during the PCR amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Sybr green kit, by Takara Bio (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Reverse transcriptase m mlv rnase h kit, by Takara Bio (1 mentions)
Sybr green rt pcr kit, by Takara Bio (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Pg lps, by Merck Group (1 mentions)
Rna extraction kit, by Promega (1 mentions)
Primescript reverse transcriptase, by Takara Bio (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Takara reverse transcription kit, by Takara Bio (1 mentions)
Sequence detector, by Bio-Rad (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Plant genomic dna kit, by Tiangen Biotech (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
7 protocols using cfx connect real time system
1
RNA Extraction and qRT-PCR Analysis
2
LPS-induced gene expression in WPMY-1 cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
WPMY-1 cells cultured with DMEM containing 0 μg/ml or 1 μg/ml P.g-LPS (SMB00610, Sigma, St. Louis, MO, USA) for 24 h. Total RNA was extracted from cells using an RNA extraction kit (Promega, Beijing, China) according to the manufacturer’s instructions and cDNA was subsequently synthesized using a TaKaRa reverse transcription kit (RR047A, Takara, Japan). qPCR was performed on the Bio-Rad CFX Connect Real-Time System using the SYBR Green kit (TaKaRa, Shiga, Japan). Amplification conditions were 95 °C for 3 min and 40 cycles each at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s. Primers sequences are shown in Additional file 1 : Table S2. Results were normalized to the housekeeping gene GAPDH and relative gene expression levels were calculated using 2^-(ΔΔCt) method.
3
Quantitative Real-Time PCR Analysis of DEGs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reverse transcription was performed using PrimeScript™ Reverse Transcriptase (TaKaRa, Dalian, China) and the product was purified using the Cycle Pure kit (OMEGA, Georgia, USA). Five upregulated and five downregulated DEGs were randomly selected. qRT-PCR primers were designed using Primer Premier 6, and their specificity was verified through PCR (Table S8 ). qRT-PCR analysis was conducted on a Bio-Rad CFX Connect™ Real-Time System using SYBR® Premix Ex Taq™ II (TaKaRa, Dalian, China). Z. japonica β-actin (GenBank accession No. GU290546) was selected as the housekeeping gene. All gene expression analyses were performed using three biological replicates. We calculated the relative expression level of genes using the 2−ΔΔCT method [10 ].
4
RNA Extraction and Real-Time PCR Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were washed twice with PBS, TRIzol reagent (Takara, Tokyo, Japan) was added to each culture dish, and the cells were scraped and collected in a 1.5 mL Eppendorf tube. The RNA extraction protocol was based on previous reports. Approximately 1 μg of RNA was reverse transcribed, and cDNA was synthesized using the Takara reverse transcription kit (Takara, Tokyo, Japan) according to the manufacturer’s instructions. qPCR was performed on the Bio-Rad CFX Connect Real-Time System using the SYBR Green kit (Takara). All PCR reactions were repeated three times. PCR amplification was performed as follows: 95°C for 3 min and 40 cycles each at 95°C for 10 s, 60°C for 20 s, and 72°C for 20 s using a Bio-Rad sequence detector. The mean normalized cycle threshold (ΔCt) was used for the statistical analysis of real-time PCR results.54 (link) All amplicon primers were designed using the NCBI primer Design Center. The primers in the study are listed in Table S1 .
5
RNA Isolation and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected and total RNA was isolated using TRIzol reagent (15596018, Invitrogen) according to the manufacturer’s instructions. Then, 800 ng of total RNA was reverse-transcribed into cDNA. Real-time quantitative PCR analysis was performed on a Bio-Rad CFX Connect Real-time System thermocycler using the SYBR Green PCR Master Mix (TaKaRa). The RNA levels were normalized using HPRT or GAPDH as an internal control. All the experiments were repeated three times. The primers are shown in detail in Supplementary Table 1 .
6
RNA Isolation and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs were isolated from the desired cells using the TRIzol (Invitrogen), reverse transcribed into cDNA using SuperScript III First-Strand Synthesis System (Invitrogen), and subjected to qRT-PCR (Bio-Rad, CFX Connect Real-Time System) with the SYBR Premix Ex Taq (TakaRa). The primers used for qPCR are listed in Table S1 .
7
Quantitative telomere length analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
This assay was essentially performed as previously described with some modifications (Cawthon 2002; (link)Vaquero-Sedas and Vega-Palas 2014) (link). First, seeds were germinated on MS medium or MS medium containing various concentrations of zeocin, and genomic DNA was extracted from the roots of 8-d-old seedlings using the Plant Genomic DNA Kit (TIANGEN, DP305). Then, the telomeric sequences were amplified using primers TelA and TelB on a Bio-Rad CFX connect Real-time System using TB Green Premix Ex Taq II (TaKaRa, RR820A). The single copy gene CYP5 was also amplified as a reference, using primers CYP5-F and CYP5-R. Two hundred nanograms of DNA per PCR was used.
The qPCR conditions were as follows: for telomeric sequences, (i) 95°C, 15 min; (ii) 2 cycles of 94°C, 15 s, and 49°C, 15 s; (iii) 32 cycles of 15 s at 94°C, 10 s at 62°C, and 15 s at 74°C; and (iv) 1 min at 72°C. For CYP5, 95°C, 10 min; 95°C, 15 s; 60°C, 1 min; and 72°C, 1 min. Totally 32 cycles. The results were calculated by 2 -ΔΔCt . Statistical significances of differences were assessed using Tukey's test.
The qPCR conditions were as follows: for telomeric sequences, (i) 95°C, 15 min; (ii) 2 cycles of 94°C, 15 s, and 49°C, 15 s; (iii) 32 cycles of 15 s at 94°C, 10 s at 62°C, and 15 s at 74°C; and (iv) 1 min at 72°C. For CYP5, 95°C, 10 min; 95°C, 15 s; 60°C, 1 min; and 72°C, 1 min. Totally 32 cycles. The results were calculated by 2 -ΔΔCt . Statistical significances of differences were assessed using Tukey's test.
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!