RNA was extracted from cells or mouse skin using the RNAeasy Plus universal kit (Qiagen 73404). RNA was quantified by absorbance at 260 nm, and its purity was evaluated by the ratios of absorbance at 260/280 nm. 2 µg of RNA was used for cDNA synthesis (Thermo Fisher 4368814, High Capacity cDNA reverse transcription kit). Quantitative real-time PCR was performed using PowerUp SYBR Green Gene Expression Assays (Thermo Fisher A25741) and a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Relative expression values were calculated using the comparative Ct (ΔΔCt) method, and transcript abundances were normalized to GAPDH transcript abundance. The primer sequences are shown in Table 1 .
Rnaeasy plus universal kit
Manufactured by Qiagen
Sourced in Germany
The RNeasy Plus Universal Kit is a product designed for the purification of total RNA from a wide range of sample types. It utilizes a guanidine-based lysis buffer and silica-membrane technology to efficiently capture and purify RNA. The kit is suitable for use with a variety of sample sources, including cultured cells, tissues, and other biological materials.
Automatically generated - may contain errors
Lab products found in correlation
Quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (2 mentions)
Truseq rna sample preparation kit v2, by Illumina (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Qiazol, by Qiagen (1 mentions)
Dnaeasy blood tissue kit, by Qiagen (1 mentions)
C1000 touch thermocycler, by Bio-Rad (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Brilliant 2 sybr green qpcr master mix, by Agilent Technologies (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
7 protocols using rnaeasy plus universal kit
1
RNA Extraction, cDNA Synthesis, and qRT-PCR
2
Tissue Isolation and RNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rats were euthanized by decapitation. Their tissues were quickly removed and snap-frozen by pre-chilled 2-methylbutane on dry ice and stored at -80°C until further use. Blood samples were collected at the time of decapitation and immediately frozen on dry ice. Brain regions, including dorsal hippocampus (dHC), anterior cingulate cortex (ACC), prelimbic/infralimic cortex (PrL/IL) and basolateral amygdala (BLA) were punched out in a cryostat via a neuropunch (19 gauge, Fine Science Tools, Foster City, CA) as described previously [22 (link)] and immediately submerged into Qiazol (Qiagen, Venlo, Netherlands). We carefully avoided contamination of choroid plexus or leptomeninges, where Igf2 mRNA is highly expressed, by avoiding taking the tissues surrounding the ventricles and immediately adjacent to leptomeninges. Choroid plexus (CP) was collected from the lateral and third ventricles. Total RNA was isolated using the RNAeasy Plus Universal Kit following the manufacture’s protocol (Qiagen, Venlo, Netherlands) and 250 ng of RNA was reverse-transcribed using QuantiTect Reverse Transcription Kit (Qiagen, Venlo, Netherlands). Genomic DNA (gDNA) was isolated from the tails using DNAeasy Blood & Tissue Kit following the manufacture’s protocol (Qiagen, Venlo, Netherlands).
3
Quantification of Osteoblast Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from uninfected and S. aureus-infected Saos-2 cells treated with 8 µg/ml RMP at various timepoints was isolated using QIAzol and RNAeasy Plus Universal Kit (Qiagen, Germany). The S. aureus-infected osteoblasts (Saos-2 + S. aureus) group was excluded from analysis due to lack of RNA. After washing with PBS, QIAzol lysis was performed on cells per 24 wells and three wells were pooled. For complementary DNA (cDNA) synthesis, 250 to 500 ng total RNA and Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland) were used. cDNA was diluted 1:5 and quantitative real-time polymerase chain reaction (qRT-PCR) was conducted in the C1000 Touch thermocycler and CFX96 Real-Time System (Bio-Rad, Germany) at an annealing temperature of 60°C in a total volume of 20 µl using 1 µl cDNA and the Brilliant II SYBR Green QPCR Master Mix (Agilent Technologies, USA). The human gene-specific forward and reverse primers with optimal amplification efficiency are summarized in Table I . For data analysis, raw cycle thresholds (Cts) were normalized to hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) reference gene (ΔCt = Cttarget – Ctreference). Fold induction to the correlating uninfected control 'Saos-2 cells + RMP' (mean of experimental replicates) was calculated using the 2−ΔΔCt method with ∆∆Ct = ∆Ctexperimental − ∆Ctcontrol.
4
RNA-seq analysis of SZ95 sebocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from SZ95 sebocyte cells using the RNAeasy Plus universal kit (Qiagen 73404). RNA quality was assessed using Agilent 2100 Bioanalyzer. Truseq RNA sample preparation kit v2 (Illumina) was used for the preparation of sequencing libraries. Sequencing was performed on an Illumina HiSeq 2500 (RRID:SCR_016383 ) for signal end 50 bp length reads. Sequence reads were mapped against the hg19 genome using TopHat. For each gene, read counts were computed using HTSeq-count and analyzed for differential expression using DESeq2.
5
Quantifying CCR6 Expression in Colorectal Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted either from tumor tissue of colorectal cancer patients or from cultured MC38, HT29 or Hct116 cells using the RNAeasy Plus Universal Kit (Qiagen, Valencia, CA). Before extracting total RNA, cell lysates were prepared from 100 mg of tumor tissue samples by mixing with 900 ml of QIAzol Lysis Reagent (Qiagen) in an Eppendorf tube followed by homogenization using the Tissue Lyser II (Qiagen). For making cell lysates from MC38, HT29 and Hct116 cells, 2×105 cells were mixed with 1 ml of the QIAzol Lysis Reagent. For cDNA synthesis, 1 µg of total RNA samples were reverse-transcribed using the SuperScript III First-Strand Synthesis System (Life Technologies) where oligo-dT was used as first synthesis primer. Semi-quantitative RT-PCR for CCR6 was performed on cDNA prepared from patient tumor tissue. The primers [28] (link) and conditions are detailed in the Methods S1 .
6
RNA-seq of Mouse Skin Transcriptome
7
Mouse Skin RNA Expression Analysis
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!