Total RNA was extracted from lysates of vaccine-stimulated PBMCs and CBMCs using the Qiagen RNeasy Minikit and DNAse treatment performed using the Qiagen RNAase Free DNAase set all per the manufacturer’s instructions. RNA concentrations were determined using the Nanodrop 1000 and cDNA generated using the Qiagen RT2 First Strand Kit. 96-well PCR array analysis was performed using the Qiagen standardized Innate and Adaptive Immune Reponses PCR Array (PAHS-0522A) and RT2 qPCR roxSYBR green kit. Web-based PCR array analyses (RT2 Profiler PCR Array Data Analysis version 3.5) was used and normalized to five reference genes (B2M, HPRT1, RPL13A, GAPDH, and ACTB). Relative quantification of gene expression was calculated by the ΔCt (relative expression × 104). Multivariate biplots of principal component analyses were performed in R 3.4.2 using ggplot2, ggord, and vegan packages using log-fold transcript abundance of gene arrays in each group. Genes were sorted using unsupervised hierarchical heatmap clustering of log-fold changes using the heatmap2 package.
Rnaase free dnaase
Manufactured by Qiagen
RNase-Free DNase is an enzyme that selectively degrades DNA, while leaving RNA intact. It is designed for use in a variety of molecular biology applications where the removal of DNA is required, such as RNA purification and sample preparation for reverse transcription and real-time PCR.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Nanodrop 1000, by Qiagen (1 mentions)
Rt2 first strand kit, by Qiagen (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Qscript cdna supermix, by Quanta Biosciences (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
3 protocols using rnaase free dnaase
1
RNA Extraction and Immune Profiling
2
RNA Extraction, Purification, and cDNA Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted using RNeasy Plus Mini kit (Qiagen; 74134) according to the manufacturer's instructions. RNAase-Free DNAase (Qiagen; 79254) was used to ensure all genomic DNA had been removed. Gel electrophoresis was used to check the integrity of the samples. The concentration of the samples was measured using a NanoDrop 3300 spectrophotometer. Based on these results, equal concentrations of RNA were converted into cDNA using SuperScript III Reverse Transcriptase (Invitrogen; 18080 044) according to the manufacturer's instructions.
3
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 from tsA-201 cells or mouse arteries was isolated and purified by applying 1 ml of TRIzol reagent per sample according to the manufacturer’s instructions (Invitrogen). RNA was purified using the RNeasy Mini kit (QIAGEN). Genomic DNA was digested using RNAase-free DNAase (1023460; QIAGEN). For cDNA synthesis, 1 µg total RNA was reverse-transcribed using the qScriptcDNA supermix (84034; Quanta Biosciences, Inc.). For real-time PCR, a cDNA amount equivalent to 15 ng initial RNA was loaded per well and amplified using PowerUP SYBR Green Mastermix (A25742; Applied Biosystems) under the following conditions: 2 min at 50°C initial UDG activation, denaturation for 2 min at 95°C, followed by 40 cycles consisting of 15 s at 95° and 60 s at 60°C. Table S1 summarizes the sets of primers designed for each gene. Fold change in the expression of mRNA for TRPV4 and AKAP150 was calculated using the 2−ΔΔCT method taking the geometric mean of the human control genes 18S, GAPDH, and α-actinin as reference in tsA-201 cells and the geometric mean of the mouse genes 18S, hypoxanthine-guanine phosphoribosyl transferase (HPRT), and β-actin in the artery samples.
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!