Total RNA was extracted from cells using an RNeasy Plus RNA extraction kit (Qiagen, 74136) according to the manufacturer’s protocol. RT-qPCR reactions were run using the QuantiTect SYBR Green RT-qPCR kit (Qiagen, 204243) on a Qiagen Rotor-Gene Q. For eRNA-qPCR, RNA was extracted using an RNeasy Plus RNA extraction kit (Qiagen, 74136) with the on-column DNAse digest, according to the manufacturer’s instructions. 500 ng of RNA was reverse-transcribed using SuperScript VILO Master Mix (Thermo Fisher Scientific, 11755250) according to the manufacturer’s instructions. eRNA levels were assessed by qPCR using a Rotor-Gene SYBR Green PCR Kit (Qiagen, 1054586) on a Qiagen Rotor-Gene Q. Relative transcript levels were determined by standard curve and normalised to the expression of RPLP0 control gene. Primers used are listed in Supplementary file 11 .
Rneasy plus rna extraction kit
Manufactured by Qiagen
Sourced in United States
The RNeasy Plus RNA extraction kit is a product designed to extract and purify total RNA from various sample types. It utilizes a silica-based membrane technology to efficiently capture and elute high-quality RNA suitable for downstream applications such as RT-PCR, northern blotting, and microarray analysis.
Automatically generated - may contain errors
Lab products found in correlation
Quantitect sybr green rt qpcr kit, by Qiagen (3 mentions)
Truseq stranded mrna library kit, by Illumina (3 mentions)
Sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Taqman reverse transcriptase kit, by Thermo Fisher Scientific (3 mentions)
Qiashredder extraction column, by Qiagen (3 mentions)
Applied biosystems quantstudio 3 real time pcr machine, by Thermo Fisher Scientific (3 mentions)
Hiseq 4000 system, by Illumina (2 mentions)
On column dnase digest, by Qiagen (2 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (2 mentions)
High capacity cdna rt kit, by Thermo Fisher Scientific (2 mentions)
Rotor gene q, by Qiagen (1 mentions)
Rotor gene sybr green pcr kit, by Qiagen (1 mentions)
Superscript vilo master mix, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Nextseq 550 platform, by Illumina (1 mentions)
19 protocols using rneasy plus rna extraction kit
1
Quantifying RNA Levels by RT-qPCR
2
RNA-Seq Protocol for Differential Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three biological replicates were sequenced per condition. Total RNA was extracted from cells using a RNeasy Plus RNA extraction kit (Qiagen, 74136). An on-column DNase digest (Qiagen, 79254) was performed according to the manufacturer’s protocol. RNA-seq libraries were generated using a TruSeq stranded mRNA library kit (Illumina, RS-122-2001) and sequenced by the University of Manchester Genomic Technologies Core Facility on a HiSeq 4000 System (Illumina).
Reads were trimmed using Trimmomatic v0.32 [56 (link)] and aligned to the human genome (GRCh37, hg19, RefSeq transcript annotation) using STAR v2.3.0 [64 (link)]. Gene expression counts were obtained using featureCounts v1.6.2 [62 (link)] and differentially expressed genes were identified using DESeq2 v1.14.1 using FDR < 0.05 [63 (link)]. For results from the lapatinib treatment timecourse, differentially expressed genes were filtered to remove genes in which no timepoint had an FPKM value > 1. Metascape [65 (link)] was used for gene ontology analysis of differentially expressed genes. Ingenuity Pathway Analysis [66 (link)] was used to predict upstream regulators.
Patient tumour samples in the OCCAMS dataset expressing ERBB2 at high levels (ERBB2HIGH) were defined by ERBB2 expression levels greater than the median +2 SD.
Reads were trimmed using Trimmomatic v0.32 [56 (link)] and aligned to the human genome (GRCh37, hg19, RefSeq transcript annotation) using STAR v2.3.0 [64 (link)]. Gene expression counts were obtained using featureCounts v1.6.2 [62 (link)] and differentially expressed genes were identified using DESeq2 v1.14.1 using FDR < 0.05 [63 (link)]. For results from the lapatinib treatment timecourse, differentially expressed genes were filtered to remove genes in which no timepoint had an FPKM value > 1. Metascape [65 (link)] was used for gene ontology analysis of differentially expressed genes. Ingenuity Pathway Analysis [66 (link)] was used to predict upstream regulators.
Patient tumour samples in the OCCAMS dataset expressing ERBB2 at high levels (ERBB2HIGH) were defined by ERBB2 expression levels greater than the median +2 SD.
3
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed once in PBS and harvested following trypsinization and centrifugation. The pelleted cells (with media removed) were frozen immediately on dry ice for at least 15 min. RNA was extracted using a QIAGEN QIAshredder extraction column (Cat# 79654) and RNeasy Plus RNA Extraction Kit (Cat# 74134). cDNA was generated using the TaqMan Reverse Transcriptase Kit according to the manufacturer’s instructions (Cat# N8080234, Applied Biosystems/Thermo Fisher Scientific, Foster City, CA). See Table S2 for qPCR primer sequences. Quantitative PCR reactions were prepared with SYBR Green Master Mix (Cat# 4367659, Life Technologies) and run on an Applied Biosystems QuantStudio 3 real-time PCR machine (Thermo Fisher). Relative transcript levels were calculated using the ΔΔCT method and normalized to the ACTB gene as described (Tarangelo et al., 2018 (link)).
4
Quantifying Gene Expression and Viral Genomes
5
RNA Extraction and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using an RNeasy Plus RNA extraction kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions, and 2 µg of total RNA was added in a reverse-transcription reaction to generate the first strand of cDNA using a High-capacity cDNA Reverse Transcription Kit (Applied Biosystem, Waltham, MA, USA). The synthesized cDNA was then subjected to qRT-PCR with the following primers (MFRN1: Forward 5′- TAG CCA ACG GGA TAG CTG G -3′; Reverse 5′- GTG GTG TAG CTC CGG TAG AAG -3′; 18S: Forward 5′- ACC CGT TGA ACC CCA TTC GTG A -3′; Reverse 5′- GCC TCA CTA AAC CAT CCA ATC GG -3′). The mRNA expression level of 18S rRNA served as an internal standard control.
6
Quantifying Gene Expression and Viral Genomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure relative gene expression by qRT-PCR, total cellular RNA was isolated using RNeasy Plus RNA extraction kit (QIAGEN). Approximately 400–2000ng RNA was normalized across samples and cDNA was generated using the High Capacity cDNA RT kit (Applied Biosystems). cDNA was then subjected to qPCR using Fast SYBR Green Master Mix (Applied Biosystems) and primers as indicated on the ViiA7 Real Time PCR system (Life Technologies). Three technical replicates were performed for each biological sample, and expression values of each replicate were normalized against GAPDH cDNA using the 2−ΔΔCt method. For relative expression (fold), control samples were centered at 1; for relative expression (%), control samples were centered at 100%. mtDNA copy number analysis was performed as described using primers specific to nuclear Tert and the D-loop region of mtDNA (listed in Extended Data Table 1 )6 (link). Relative HSV-1 genome abundance was determined using primers specific for nuclear Tert and HSV-1 UL30 or TK. Relative MHV68 genome abundance was determined using primers specific for nuclear Tert and MHV68 ORF40. Relative Vaccinia genome abundance was determined using primers specific for nuclear Tert and VV DNApol E9L.
7
Quantitative Gene Expression Analysis
8
FMDV Replicon Genome Amplification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following cotransfection of BHK-21 cells with mCherry and ptGFP in vitro-transcribed replicon RNA, cells were detached by trypsin and washed once in ice-cold phosphate-buffered saline (PBS), and total RNA was extracted using the RNeasy Plus RNA extraction kit (Qiagen) according to the manufacturer's protocol. Total RNA was treated on-column with RQ1-DNase, and total FMDV cDNA was amplified using Superscript II (Life Technologies) according to the manufacturer's protocol. Both mCherry and ptGFP replicon genomes were amplified separately using forward primers specific for either mCherry (mCherry_fwd) or ptGFP (ptGFP_fwd) with FMDV-specific reverse primer (FMDV_7200_7180) with Phusion High-Fidelity DNA polymerase (NEB). PCR products were blunt-end ligated into pCRBlunt. Individual colonies were isolated, and the presence of insertional mutation was determined by DNA sequencing.
9
RNA Extraction and RT-qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cells using a RNeasy Plus RNA extraction kit (Qiagen, 74136) according to the manufacturer’s protocol. RT-qPCR reactions were run using a QuantiTect SYBR Green RT-qPCR kit (Qiagen, 204243) on a Qiagen Rotorgene Q. Relative copy number of transcripts was determined from a standard curve and then normalised by the expression of RPLP0 control gene. Primer pairs are listed in Supplementary Table 5 .
10
Quantifying msl2 and roX2 Transcript Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine transcript abundance of msl2 and roX2 following RNAi treatment, 500μL of cells were collected following the 6-day incubation period. After pelleting cells and removing the media, total RNA was extracted using the RNeasy Plus RNA extraction kit (Qiagen). A total of 1μg of RNA was reverse-transcribed to cDNA using the SuperScript Vilo cDNA Synthesis kit (ThermoFisher Scientific) by following the manufacturer’s instructions. Targets were amplified from cDNA using validated primers at a concentration of 200nM. Primer sequences for qRT-PCR are available upon request. Three technical replicates for each sample were amplified using SYBR Green on an Applied Biosystems StepOnePlus™ Real-Time PCR System. The obtained relative abundance values were averaged and used to calculate ΔCt relative to PKA as an internal control.
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!