Total RNA was isolated from seedlings frozen in liquid nitrogen with TRIZOL reagent (Invitrogen) according to the manufacturer’s protocol. Concentration of RNA was determined by measuring OD at 260 nm. First-strand cDNA was synthesized with the SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen). Real-time quantitative PCR (qRT-PCR) analysis was performed using the LightCycler Quick System 350S (Roche Diagnostics K.K.) with SYBR Premix Ex Taq (Takara Bio, Inc.). Each PCR reaction contained 1 × SYBR Premix Ex Taq, 0.2 μM of each primer, and 2 μL of a 1:10 dilution of the cDNA in a final volume of 20 μL. The PCR programme included: the initial denaturation, at 95°C, for 30 s; PCR, of 40 cycles at 95°C, for 5 s, and then at 60°C, for 20 s with a temperature transition rate of 20°C s-1. In melting curve analysis, PCR reactions were denatured at 95°C, annealed at 65°C, then a monitored release of intercalator from PCR products or primer dimmers by an increase to 95°C with a temperature transition rate of 0.1°C s-1. Standard curves were created using PCR products by 10-fold serial dilutions. The MPK3 and MPK6 genes expression profiles were normalized using actin mRNA as an internal control. The primers for Real-time PCR are listed in S1 Table .
Lightcycler quick system 350s
Manufactured by Roche
Sourced in Germany, Japan
The LightCycler Quick System 350S is a real-time PCR instrument designed for rapid and efficient nucleic acid amplification and detection. The system provides accurate and reliable quantification of target sequences in a variety of sample types. Its core function is to perform real-time PCR analysis with a focus on speed and ease of use.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Thunderbird sybr qpcr mix, by Toyobo (5 mentions)
Quantitect reverse transcription kit, by Qiagen (4 mentions)
Sybr premix ex taq, by Takara Bio (3 mentions)
Revertra ace α, by Toyobo (3 mentions)
Superscript 2 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Sybr green real time pcr master mix plus, by Toyobo (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Extract n amp plant pcr kit, by Merck Group (1 mentions)
Genelute mammalian total rna miniprep kit, by Merck Group (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Isogen, by Fujifilm (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
12 protocols using lightcycler quick system 350s
1
Quantitative Real-Time PCR Analysis of Gene Expression
2
Retinal Total RNA Extraction and RT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA of the retina was extracted using TRIzol (Invitrogen, CA, USA). The extracts were reverse-transcribed using random primers and a QuantiTect Reverse Transcription kit (QIAGEN, Tokyo, Japan), according to the manufacturer’s instructions. Real-time RT-PCR was performed using a LightCycler Quick System 350S (Roche Diagnostics, Tokyo, Japan), with SybrGreen Realtime PCR Master Mix Plus (Toyobo, Osaka, Japan). The specific primers used in the present study are listed in Table 1 .
3
Endophyte-Infected Ryegrass DNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Extraction of genomic DNA of perennial ryegrass, infected with endophyte strains was performed using Extract-N-Amp Plant PCR kit (Sigma). Ten mg of plant tissue was ground using an electric homogenizer (BioMasher, Nippi), suspended in 150 μl of extraction buffer and centrifuged at 12,000 xg for 1 min. Total DNA was purified from the supernatant (100 μl) according the manufacturer’s instructions. Quantitative PCR was performed using LightCycler Quick System 350S (Roche Applied Science) with Thunderbird SYBR qPCR Mix (Toyobo). Gene-specific primers used for expression analysis are listed in S4 Table .
4
In vivo NF-κB Decoy Transfection Modulates Tumor-Associated Macrophage Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 4 days after inoculation of EAC cells into mice, we carried out in vivo NF-κB decoy transfection. After 24 h of in vivo NF-κB decoy transfection, the ascites were harvested, and TAMs were separated from ascites. Total RNA was isolated from TAMs using a GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA). Reverse transcription of mRNA was carried out using a PrimeScript RT reagent Kit (Takara Bio, Shiga, Japan). Detection of cDNAs (IL-10, IL-12p70, TNF-α, IL-6, VEGF-A, and GAPDH) was carried out by real-time PCR using SYBR Premix Ex Taq (Takara Bio) and a LightCycler Quick System 350S (Roche Diagnostics, Indianapolis, IN, USA). The primers for IL-10, IL-12, TNF-α, IL-6, and GAPDH cDNAs were as follows: IL-10, 5′-GCT CTT ACT GAC TGG CAT GAG-3′ (forward) and 5′-CGC AGC TCT AGG AGC ATG TG-3′ (reverse); IL-12, 5′-ACT CTG CGC CAG AAA CCT C-3′ (forward) and 5′-CAC CCT GTT GAT GGT CAC GAC-3′ (reverse); TNF-α, 5′-CCT CCC TCT CAT CAG TTC TA-3′ (forward) and 5′-ACT TGG TGG TTT GCT ACG AC-3′ (reverse); IL-6, 5′-TAG TCC TTC CTA CCC CAA TTT CC-3′ (forward) and 5′-TTG GTC CTT AGC CAC TCC TTC-3′ (reverse); VEGF-A, 5′- AGC ACA GCA GAT GTG AAT GC-3′ (forward) and 5′-AAT GCT TTC TCC GCT CTG AA-3′ (reverse); and GAPDH, 5′-TCT CCT GCG ACT TCA ACA-3′ (forward) and 5′-GCT GTA GCC GTA TTC ATT GT-3′ (reverse).
5
Transcriptome Analysis of N. benthamiana
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs were isolated from N. benthamiana leaves using TRIzol Reagent (Life Technologies, United States) and cDNA synthesis was conducted using ReverTra Ace-α- (Toyobo, Japan) according to the manufacturer’s instructions. Quantitative RT-PCR (qRT-PCR) analysis was performed using LightCycler Quick System 350S (Roche Applied Science, Germany) with Thunderbird SYBR qPCR Mix (Toyobo, Japan). Expression of N. benthamiana EF-1α gene was used as an internal standard. Gene-specific primers used for expression analysis were listed in Supplementary Table S4 .
6
Quantitative RT-PCR Analysis of AOX1a Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from 2.5-week-old plant seedlings frozen in liquid nitrogen with TRIZOL reagent (Invitrogen) according to the manufacturer’s protocol. Concentration of RNA was determined by measuring OD at 260 nm. First-strand cDNA was synthesized with the SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen). For real-time quantitative PCR (qRT-PCR) analysis, it was performed using the LightCycler Quick System 350S (Roche Diagnostics K.K.) with SYBR Premix Ex Taq (Takara Bio, Inc.). Each PCR reaction contained 1 × SYBR Premix Ex Taq, 0.2 μM of each primer, and 2 μL of a 1:10 dilution of the cDNA in a final volume of 20 μL. The PCR programme was used: initial denaturation, 95°C, 30 s; PCR, 40 cycles of 95°C, 5 s, 60°C, 20 s with a temperature transition rate of 20°C s-1. In melting curve analysis, PCR reactions were denatured at 95°C, annealed at 65°C, then a monitored release of intercalator from PCR products or primer dimmers by an increase to 95°C with a temperature transition rate of 0.1°C s-1. Standard curves were created using PCR products by 10-fold serial dilutions. The AOX1a gene expression profile was normalized using actin mRNA as an internal control. The primers for real-time quantitative PCR are listed in S1 Table .
7
RNA Isolation and qRT-PCR Analysis in N. benthamiana
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs were isolated from N. benthamiana leaves using TRIzol Reagent (Thermo Fisher Scientific) and cDNA synthesis was conducted using ReverTra Ace-α- (Toyobo, Osaka, Japan). Quantitative RT-PCR (qRT-PCR) analysis was performed using LightCycler Quick System 350S (Roche Applied Science, Penzberg, Germany) with Thunderbird SYBR qPCR Mix (Toyobo). The expression of N. benthamiana EF-1α gene was used as an internal standard. Gene-specific primers used for expression analysis were listed in Supplementary Table 2 .
8
Quantifying RNA Expression in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from isolated macrophages, hepatocytes, and Hepa1–6 cells using an RNeasy Micro Kit (Qiagen, Hilden, Germany), and from the liver using an RNeasy Mini Kit (Qiagen). cDNA synthesis was performed with a QuantiTect Reverse Transcription Kit (Qiagen) using 100 ng of RNA from cultured cells and 1 µg of total RNA from the liver. Real-time PCR was performed using the Light Cycler Quick System 350S (Roche Diagnostic, Mannheim, Germany)45 (link). The relative amount of mRNA was calculated with β-actin mRNA serving as the invariant control. The oligonucleotide primers are presented in Supplementary Table 1 .
9
Olfactory Receptor Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated from isolated olfactory epithelium, isolated islets, and MIN6 cells using the RNeasy Mini Kit or the RNeasy Micro Kit (Qiagen, Valencia, CA, USA). RNA was reverse transcribed into cDNAs using a QuantiTect Reverse Transcription Kit (Qiagen). Olfr15, Olfr821, Olfr1222, Olr1356, OR2C1, rat and human β-actin, Gαolf, Omp and murine β-actin, were detected by RT-PCR, with the oligonucleotides presented in Supplementary Table S2 .
On the other hand, template cDNAs were evaluated with a real-time PCR quantitative system (Light Cycler Quick System 350 S; Roche Diagnostics, Mannheim, Germany), with the oligonucleotides presented in Supplementary TableS3 . The relative amount of mRNA was calculated with β-actin mRNA as the invariant control.
On the other hand, template cDNAs were evaluated with a real-time PCR quantitative system (Light Cycler Quick System 350 S; Roche Diagnostics, Mannheim, Germany), with the oligonucleotides presented in Supplementary Table
10
Quantifying mRNA Levels from Mouse Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from 50 mg of mouse liver, epididymal WAT, brown adipose tissue, cardiac and skeletal muscles with Isogen (Wako Pure Chemical), and cDNA was synthesized with a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics Gmbh, Mannheim, Germany) using 5 μg of total RNA. cDNA synthesized from total RNA was evaluated with a real-time PCR quantitative system3 (link) (Light Cycler Quick System 350S; Roche Diagnostics Gmbh). The relative amount of mRNA was calculated with 28SrRNA or β-actin mRNA as the invariant control. The primers used are described in the Supplementary Table 2 .
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!