The real-time fluorescence quantitative PCR (FQ-PCR) procedure was in accordance with previous reports [27 (link)]. It was performed with an Mx3005P real-time qPCR system (Agilent, California, USA) and Ultra SYBR Mixture (Cwbiotech, Beijing, China), using a 20-μl reaction solution containing 10 μl 2× Ultra SYBR Mixture (Cwbiotech, Bengjing, China), 0.5 μl of each primer (Table 1 ), 2 μl DNA template and 7 μl DNase/RNase-free water. The thermal cycler profile was 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 30 s. All samples were measured in triplicate. Differences in gene expression were calculated using the 2-cycle threshold method. β-actin was used as a reference gene. For analysis of mRNA expression of each validated gene, raw data were normalized against the values obtained for β-actin mRNA, and the fold changes in the expression of each gene in the infected group vs. the control group were determined. The standard curve for PCV2 was obtained using 10-fold dilutions of plasmid DNA for viral loads.
Ultrasybr mixture
Manufactured by CWBIO
Sourced in China, United States, Switzerland, Germany
The UltraSYBR Mixture is a real-time PCR reagent designed for sensitive and specific detection of target DNA sequences. It contains all the necessary components for the amplification and detection of DNA, including a proprietary fluorescent dye that binds to double-stranded DNA.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (130 mentions)
Hifiscript cdna synthesis kit, by CWBIO (66 mentions)
Trizol, by Thermo Fisher Scientific (36 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (30 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (25 mentions)
Primescript rt reagent kit, by Takara Bio (25 mentions)
Ultrapure rna kit, by CWBIO (24 mentions)
Trizol reagent, by CWBIO (19 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (16 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (11 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (10 mentions)
Lightcycler 96, by Roche (9 mentions)
Lightcycler 480 2, by Roche (9 mentions)
Cw2569, by CWBIO (9 mentions)
Rnaiso plus, by Takara Bio (8 mentions)
Trizol reagent, by Takara Bio (8 mentions)
Trizol, by CWBIO (7 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (7 mentions)
Revertra ace qpcr rt kit, by Toyobo (6 mentions)
Revertra ace qpcr kit, by Toyobo (6 mentions)
404 protocols using ultrasybr mixture
1
Real-Time Fluorescence Quantitative PCR Assay
2
Quantitative RT-PCR Analysis of snRNAs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted with the TRIzol reagent and reverse−transcribed into complementary DNA (cDNA) using the Takara PrimeScript RT reagent Kit (Takara Bio, Kusatsu, Japan) in 20 μl reaction according to the manufacturer’s instructions. The qRT-PCR was performed using UltraSYBR Mixture (CWBio, Beijing, China) in a volume of 25 μl (12.5 μl of 2 × UltraSYBR Mixture, 0.5 μl of forward premier, 0.5 μl of reverse premier, 1 μl cDNA and 10.5 μl water). The reactions were performed with LightCycler 480 qPCR system (Roche Diagnostics, Germany). 18S acted as control as described previously (18 (link)). The relative expression of snRNAs was evaluated by the comparative cycle threshold (ΔCt) method: (ΔCt = CtsnRNA – Ct18S) as described previously (19 (link)). The qPCR primers are listed in Table 1 .
3
Validating Soybean Transcriptome via qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To validate the transcriptome data, the expression of 20 genes was evaluated by quantitative real-time PCR (qRT-PCR) analysis. cDNAs were synthesized according to the manufacturer’s protocol (Takara, Dalian, China) and used as template for qRT-PCR analysis using primers based on the reference soybean gene sequences (S1 Table ). qRT-PCR was conducted using UltraSYBR mixture (CWBIO, China) in a typical 20μl PCR mixture that included 10μl of UltraSYBR mixture, 2μl (100 ng) of template cDNA, and 0.4μM of each PCR primer. Cycling conditions were 95°C for 2 min, followed by 40 cycles of 95°C for 10 s (denaturation), followed by 60°C for 20 s (annealing and extension). The melting curve of each PCR amplicon was obtained under the following conditions: 95°C for 10 s followed by a constant increase in temperature from 65 to 95°C at an increment of 0.5°C / cycle. Samples were run on the StepOnePlus Real-Time PCR System (ABI, USA). Relative expression of the target genes was analyzed with the 2-ΔΔCt method using ABCT,CONS4,ACT11 as internal controls [32 ]. All samples were amplified with three biological replications and with three technical replication each biological replication.
4
Quantitative RT-PCR Analysis using UltraSYBR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RT-qPCR analysis was performed using UltraSYBR Mixture (cwbiotech, Jiangsu, China), and the reactions were conducted in a final volume of 25 µl containing 12.5 µl of 2x UltraSYBR Mixture, 0.2 uM of each primer, 1.0 µl of diluted cDNA and nuclease-free water. A Bio-Rad CFX96 system (Bio-Rad, Hercules, CA, USA) was used to perform RT-qPCR reaction with the following conditions: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, and a melting-curve program was generated by the instrument. Each real-time PCR reaction was performed with three technical and biological replicates.
5
Quantitative Real-Time PCR Analysis of Tilapia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primers designed based on the O. niloticus transcriptome genome sequences are presented in Table 2 . The primer amplification efficiency of all genes was between 90% and 110%. Total RNA was extracted by TRIzol® reagent (RN0101, Invitrogen, Shanghai, China). The quantity and concentration of total RNA were measured by the Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, NC, USA). The first-strand cDNA synthesis was performed using the PrimeScriptTM RT Reagent kit (KR116, Tiangen Biotech, Beijing, China).
The reaction volume used in qRT-PCR was 20 μL containing 10 μL 2 × Ultra SYBR Mixture (CWbio, Nanjing, China), 1.6 μL of each forward and reverse primers (2.5 μmol/μL), 1 μL of diluted cDNA (200 ng/μL) and 7.4 μL of RNAase free water. All procedures are performed according to the manufacturer’s instruction. β-actin was used as the reference gene. qRT-PCR data were analyzed with the 2−ΔΔCt method.
The reaction volume used in qRT-PCR was 20 μL containing 10 μL 2 × Ultra SYBR Mixture (CWbio, Nanjing, China), 1.6 μL of each forward and reverse primers (2.5 μmol/μL), 1 μL of diluted cDNA (200 ng/μL) and 7.4 μL of RNAase free water. All procedures are performed according to the manufacturer’s instruction. β-actin was used as the reference gene. qRT-PCR data were analyzed with the 2−ΔΔCt method.
6
Ileum Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ileum samples from the control group, low Cd treatment and antibiotic treatment were collected to extract total RNA using an Ultrapure RNA kit (CWbio, Beijing, China) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized by reverse transcription using the EasyQuick RT MasterMix kit (CWbio, Beijing, China). The expression of zonula occludens 1 (ZO-1), junctional adhesion molecule A (JAM-A), occluding was quantified by quantitative polymerase chain reaction (qPCR). Briefly, the primers used to amply these genes are list in Table S1 , qPCR was performed on LightCycler 96 (Roche, Santa Clara, CA, USA) in a 50-μL reaction mixture containing 25 μL of 2× UltraSYBR Mixture (CWbio, Beijing, China), 0.3 μM of each primer, and cDNA as a template. The ratio of gene expression was recorded as the fold change in expression between the treated samples and the control group. The results were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene. Each sample was run in triplicate.
7
Quantifying miRNA Targets in Skeletal Muscle
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five miRNAs (miR-181a, mir-199, mir-127, mir-133, and mir-1) were selected from differentially expressed miRNAs that were negatively correlated with their targeted genes and their targeted genes (calsequestrin 1 [CASQ1], muscleblind-like splicing regulator 1 [MBNL1], carbonic anhydrase 4 [CA4], insulin-like growth factor 2 [IGF2], and myelin expression factor 2 [MYEF2]) were characterized and quantified by quantitative PCR (qPCR). The cDNA was synthesized by reverse transcription for further experiments. We performed qPCR with each sample repeated three times. Quantitative real-time (RT)-PCR was performed as follows: 1 ng of cDNA was added to reaction mixture containing 5 μL 2×UltraSYBR Mixture (CWBIO, Beijing, China), 0.6 μL primers and water added to final volume of 10 μL. The miRNA detection program was 95°C for 10 min, followed by 45 cycles of 95°C for 25 s, 58°C for 10 s, 72°C for 10 s. The β-actin gene was used as reference gene. Genes expression levels were evaluated as 2−ΔΔct. The primers used in this experiment were shown in Supplementary Table S1 .
8
Transcriptional Profiling of Melanoma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from melanoma cells using TRIzol reagent (CWBIO, Beijing, China) according to the manufacturer's instructions. First-strand cDNA was synthesized from 1 μg of total RNA using a PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time, Takara Bio Inc., Tokyo, Japan). The 25-μl reaction mixture included 2 μl cDNA (50 μg/μl), 1 μl of each primer (Genewiz Biological Technology Co., Ltd), 12.5 μl 2× UltraSYBR Mixture (CWBIO), and 8.5 μl ddH2O. PCR was performed according to the manufacturer's instructions using the Rotor Gene 3000 real-time system (Corbett Robotics Inc., San Francisco, CA, USA) with the following cycling conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 5 sec and 60°C for 1 min. GAPDH was used as an internal control. Relative quantification wasperformed using the 2-ΔΔCt method. Primer sequences for the target genes are listed in Table I .
9
Quantifying ECM-receptor Interaction Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nine genes, which were included in both profile 5 and belonged to extracellular matrix (ECM)-receptor interaction and focal adhesion pathway, were selected. Total RNA was extracted from the hippocampus using a standard TRIzol procedure from each tissue sample (Cat#CW0581, CWbio., Beijing, China). Total RNA was purified (Cat#CW2090, CWbio., Beijing, China), and cDNA synthesis was performed using a HiFi-MMLV cDNA reagent kit (Cat#CW0744, CWbio., Beijing, China). Equivalent amounts of cDNA were used for real-time PCR in a 20-μL reaction volume containing 10 μL of 2×UltraSYBR Mixture (Cat#CW0956, CWbio., Beijing, China) and 2 μL of specific primer pairs. The qRT-PCR reaction was incubated at 95°C for 15 min, followed by 45 cycles of 95°C for 10 s, 55°C for 20 s, and 72°C for 20 s, and a melt curve analysis. Each qRT-PCR was conducted in triplicate performed in a CFX96 Touch (Bio-Rad, CA, USA). Sequences of each primer are listed in Supplementary Table S1. The real-time value for each sample was averaged and compared using the cycle threshold method, where the amount of target RNA (2−ΔΔCT) was normalized to the endogenous monkey actin reference (ΔCT).
10
Quantitative PCR 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 uteri and marrow derived dendritic cells (BMDCs) with Trizol® reagent (Invitrogen, Carlsbad, CA) and was reverse transcribed into stable cDNA using M‐MLV reverse transcriptase buffer (Invitrogen, Carlsbad, CA) in accordance with the manufacturer's instructions. The amplification of cDNA was performed using 2× UltraSYBR Mixture (CWBIO, Co. Ltd, Beijing, China). Real‐time quantitative PCR was carried out with a LightCycler 480 PCR instrument (Roche, Indianapolis, IN). The target gene mRNA expression was normalized to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) expression. The fold change was calculated as 2−ΔΔCt (cycle threshold). The primers used for quantitative PCR are listed in Table S1 .
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!