In vivo tissue was homogenized in RLT Buffer (Qiagen) using a Bullet Blender (Next Advance, Averill Park, NY) according to the manufacturer’s protocol. Organoids (see later) were lysed directly into RLT Buffer and vortexed until the solution was homogenous. RNA was purified using the RNEasy Mini RNA purification kit (Qiagen), and RNA was then transcribed to cDNA using the iScript RT Supermix (Bio-Rad, Mississauga, Ontario, Canada) according to the manufacturer’s protocols.
Rneasy rna purification mini kit
Manufactured by Qiagen
Sourced in Germany
The RNeasy RNA purification mini kit is a laboratory tool designed to extract and purify RNA from a variety of sample types. It utilizes a silica-membrane-based technology to efficiently capture and isolate RNA molecules for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Qscript cdna synthesis kit, by Quanta Biosciences (6 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Nk isolation kit 2, by Miltenyi Biotec (3 mentions)
Easysep mouse nk cell isolation kit, by STEMCELL (2 mentions)
Rnase free dnase, by Qiagen (2 mentions)
Icycler, by Bio-Rad (2 mentions)
430 2.0 mouse expression arrays, by Thermo Fisher Scientific (2 mentions)
Affymetrix expression console v1, by Thermo Fisher Scientific (2 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (2 mentions)
Rnase free dnase set, by Qiagen (2 mentions)
Rlt buffer, by Qiagen (1 mentions)
Iscript rt supermix, by Bio-Rad (1 mentions)
Bullet blender, by Next Advance (1 mentions)
Rna extraction kit, by Takara Bio (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Beadarray system, by Illumina (1 mentions)
Rt2 sybr green rox qpcr mastermix, by Qiagen (1 mentions)
Rt2 first strand kit, by Qiagen (1 mentions)
Rnase free dnase kit, by Qiagen (1 mentions)
21 protocols using rneasy rna purification mini kit
1
RNA Extraction and cDNA Synthesis
2
RNA Extraction and Reverse Transcription Protocol
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Total RNA was extracted from the infected and negative control embryo fibroblast cells harvested at each time point using the RNeasy Mini RNA Purification Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Viral RNA was extracted from culture supernatants by using an RNA extraction kit (Takara, Japan). RNA in each sample was quantified using an Ultrospec 2000 mass spectrophotometer (Pharmacia Biotech, Uppsala, Sweden). Approximately 2 ug RNA from each sample was treated with DNase to remove genomic DNA and was later reverse-transcribed to cDNA using the SuperScript® III First-Strand Synthesis System (Clontech) according to the manufacturer’s protocol.
3
Transcriptomic Analysis of Insect Tissues
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Whole RNA from fat body, hematopoietic organ, midgut, and hemocytes was isolated with an RNeasy Mini RNA purification kit (Qiagen, Hilden, Germany). Pooled tissue from four animals was used for each isolation. The RNA quality, purity, and concentration were evaluated spectrophotometrically with a NanoDrop 1000 (Thermo Fisher, Waltham, USA) and denaturing RNA electrophoresis. Prior to reverse transcription, the contaminating gDNA was removed by DNAse I digestion (DNAse I RNAse free, Thermo Fisher, Waltham, USA). 550 ng purified RNA of each sample was transcribed to cDNA with SuperScript III Reverse Transcriptase (Life Technologies, Carlsbad, USA) using oligo(dT)20 primers.
4
mRNA Purification and Microarray Analysis
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The RNeasy mini RNA purification kit (Qiagen, Manchester, UK) was used to purify mRNA from the RKO parental cell line and the irinotecan-resistant clones RSC316 and RSC526 according to the manufacturers details. The mRNA quality was verified by measuring 260/280 and 260/230 ratios using a Nanodrop (Wilmington, USA). The mRNA samples were subsequently sent to Cambridge Genomic Services (CGS, Cambridge, UK) for additional quality control prior to microarray analysis using a HumanHT-12 v4 WG-GX Beadchip on the Illumina BeadArray system. The data was returned in the format X and the real-fold change in mRNA levels calculated using the equation 2X.
5
Quantifying CT-RCC HERV-E Expression
6
RNA Extraction from Human Liver
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RNA was extracted from human liver specimens using the RNeasy Mini RNA purification kit (Qiagen GmbH, Hilden, Germany). RNA extracted from human-derived cells and generated HCC cell lines using the RNeasy RNA Micro purification kit (Qiagen GmbH, Hilden, Germany).
7
Quantitative Analysis of Cytokine Expression
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Total RNA was extracted from kidneys' tissue from infected groups at 7 dpi using an RNeasy Mini RNA Purification Kit and RNase-Free DNase Kit (QIAGEN) for RNA purification according to the manufacturer's instructions. The expression of different cytokines was quantified by reverse transcription real-time PCR (RT-PCR) using relative quantification. The primers and probes used in this study have been described previously: IL-6, IFN-γ and 28srRNA (13) and have been described in table 1.
Quantitative real-time RT-PCR was performed using Quantitect probe RT-PCR (QIAGEN) according to the manufacturer's recommendations. Real-Time RT-PCR was carried out using a 7500 Real-time PCR System (Applied Biosystems). PCR conditions were the same for each cytokine gene, as follows: 30 min at 50 C, 95 _C for 15 min, followed by 40 cycles of 95 _C for 15 sand 60 _C for 1 min.
Statistical analysis for fold changes in cytokine levels were determined by the ΔΔCt method (Livak and Schmittgen, 2001) (link), using 28S ribosomal RNA as the endogenous reference gene to normalize the level of the target gene expression.
Quantitative real-time RT-PCR was performed using Quantitect probe RT-PCR (QIAGEN) according to the manufacturer's recommendations. Real-Time RT-PCR was carried out using a 7500 Real-time PCR System (Applied Biosystems). PCR conditions were the same for each cytokine gene, as follows: 30 min at 50 C, 95 _C for 15 min, followed by 40 cycles of 95 _C for 15 sand 60 _C for 1 min.
Statistical analysis for fold changes in cytokine levels were determined by the ΔΔCt method (Livak and Schmittgen, 2001) (link), using 28S ribosomal RNA as the endogenous reference gene to normalize the level of the target gene expression.
8
RNA Extraction and cDNA Synthesis
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Total RNA was isolated from mouse tissues, and cell lines using RNeasy mini RNA purification kit (Qiagen) and DNase-treated using the Ambion DNA-free DNase kit (Thermo Fisher Scientific) according to the manufacturer's instructions. cDNA was synthesised from 500 ng RNA in a 10 μL reverse transcriptase reaction using iScript Reverse Transcription Supermix kit for RT-qPCR (Bio-Rad) .
9
NK Cell Isolation and Gene Expression
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Cultured NK cells were purified using MACS purification EasySep™ Mouse NK cell isolation kit (Stemcell technologies, Vancouver, Canada), prior to stimulation. RNA was isolated using the RNeasy RNA purification mini kit (QIAGEN, Hilden, Germany), according to manufacturer’s protocol. From purified RNA, cDNA was synthesized using the reverse-transcriptase kit qScript cDNA synthesis kit (Quanta Biosciences, Beverly, MA, USA). Real time PCR was performed in triplicates in a 96-well plate using iQ SYBR Green-based detection on an ABI 7900HT fast qPCR machine. For the analysis of mRNA levels the derived values were normalized to the average of values obtained from the mRNA analysis of 3 different house-keeping genes- RPLP0, GAPDH, and HPRT.
10
Quantitative PCR for Gene Expression
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RNA was purified using the RNeasy RNA purification Mini Kit (Qiagen). Genomic DNA was digested with RNase-free DNase (Qiagen) following the manufacturer's instructions and reverse-transcribed using the iScript cDNA synthesis kit (BioRad). Quantitative PCR was performed in a 384-well plate format using Power SYBR Green PCR master Mix (Applied Biosystems) on an Applied Biosystems Real-Time PCR instrument. GAPDH mRNA was used for normalization.
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