RNA was extracted from rodent left ventricles using the RNeasy Maxi Kit (Qiagen, Crawley, UK). Total RNA (2 µg) was reverse-transcribed using a Taqman reverse transcriptase kit. cDNA (20 ng) was added to each well of a polymerase chain reaction array for quantitative polymerase chain reaction performed (Masterplex® realplex thermal cycler; Eppendorf, Germany). The comparative Ct method (2-∆∆Ct) was used, with hypoxanthine-guanine phosphoribosyltransferase (HPRT) or cyclophilin as the housekeeping gene. For samples obtained from sham-operated and SAD animals, evaluation of (2-∆∆Ct) was defined as the fold change in gene expression relative to the rat cerebral cortex. Primers are detailed in Supplementary Table 4 .
Rneasy maxi kit
Manufactured by Qiagen
Sourced in United States, Germany, United Kingdom
The RNeasy Maxi Kit is a laboratory equipment product designed for the purification of total RNA from a wide variety of sample types. It utilizes a silica-membrane technology to efficiently capture and purify RNA molecules, enabling their subsequent use in various downstream applications.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Lightcycler 480 real time pcr system, by Roche (9 mentions)
Miscript sybr green pcr kit, by Qiagen (8 mentions)
Miscript reverse transcription kit, by Qiagen (5 mentions)
Miscript 2 rt kit, by Qiagen (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Hiseq 2000, by Illumina (4 mentions)
Rneasy minelute kit, by Qiagen (4 mentions)
Turbo dnase, by Thermo Fisher Scientific (3 mentions)
Nanodrop, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (2 mentions)
Abi 7900 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
E gel ex, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 2000c, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Dneasy kit, by Qiagen (2 mentions)
68 protocols using rneasy maxi kit
1
Quantitative PCR Analysis of Gene Expression
2
Extraction of HuRNA from PBMCs
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HuRNA was extracted from peripheral blood mononuclear cells (PBMCs) (from buffy coats, see below) by using the RNeasy Maxi Kit Qiagen, Cat. No. 75162, Dusseldorf, Germany). The resulting RNA was controlled by 2% agarose gel electrophoresis.
3
Transcriptomic Analyses of Snail Populations
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Snails for transcriptome analyses were originally collected from different sites in France (MOTU3), Switzerland (MOTU5) and Germany (R. auricularia) between 2005 and 2011 (Additional file 6 ) and kept in water basins untill the start of this project. Information on treatment and sequence generation for R. balthica snails is reported in a previous study and documented in Feldmeyer et al. [30 (link)]. We placed individual snails in different temperature treatments. In 2011 snails were transferred to 500 ml glass jars and underwent one of six treatments, with two individuals per population, per treatment: Three days at 10 °C or 30 °C with and without aeration, 10 min temperature shock at either 4 °C or 36 °C before storage in RNAlater (Qiagen). RNA was isolated following the RNeasy maxi kit protocol (Qiagen). cDNA production, normalization, and sequencing was performed by GenXPro GmbH (Frankfurt, Germany). Due to technical issues and different time points of sequencing, the three libraries were sequenced on different platforms, MOTU3 + R. auricularia on the 454 FLX system, and MOTU5 on an Illumina HiSeq 2000. Since we were interested in sequence divergence between species, the usage of different platforms will not impair data analyses as might be expected for gene expression studies.
4
Quantification of miR-612 and NOB1 Expression
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Total RNA was extracted from tissues and cultured cells using TRIzol reagent (Invitrogen), followed by purification using an RNeasy Maxi kit (Qiagen). MiR‐612 expression was examined using the TaqMan microRNA Assay Kit (Thermo Fisher Scientific, Inc) in an ABI 7900 real‐time PCR system (Thermo Fisher Scientific, Inc) following the prescribed protocols. NOB1 mRNA expression was detected as described in our previous study.18 The endogenous control for miR‐612 was U6 while that for NOB1 was GAPDH. Relative expression levels were calculated by the 2−ΔΔCt method using ABI software.19
5
RNA-Seq Analysis of E16.5 Mouse Forebrain
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Total RNA was extracted from two replicates of E16.5 mouse forebrain tissue and purified using
the RNeasy Maxi Kit (Qiagen, Venlo, Limburg, The Netherlands) and sent to Otogenetics for
ribosomal RNA depletion, cDNA production using random primers, library preparation, and pair end
sequencing (~100 bp) using Illumina HiSeq2000. Resulting reads were demultiplexed and aligned
to the mouse genome (mm9) using TopHat v2.0.7.49 (link),51 (link),52 (link) The two replicates were merged and
read counts mapping to each transcript were obtained using HTseq.53 (link) Expression of each transcript was quantified as fragments per kilobase of
transcript per million fragments mapped (FPKM) by dividing the total number of reads mapping to each
transcript by transcript length, and the total number of reads aligned to the genome divided by one
million. The Wilcoxon test from the statistical toolkit R was used to calculate differences in gene
expression between genes whose promoter contains an Auts2-marked site and all other genes. RNA-seq
data from this study are available in SRA (http://www.ncbi.nlm.nih.gov/sra ; SRR experiment SRR1298758 (replicate 1) and
SRR1298760 (replicate 2)).
the RNeasy Maxi Kit (Qiagen, Venlo, Limburg, The Netherlands) and sent to Otogenetics for
ribosomal RNA depletion, cDNA production using random primers, library preparation, and pair end
sequencing (~100 bp) using Illumina HiSeq2000. Resulting reads were demultiplexed and aligned
to the mouse genome (mm9) using TopHat v2.0.7.49 (link),51 (link),52 (link) The two replicates were merged and
read counts mapping to each transcript were obtained using HTseq.53 (link) Expression of each transcript was quantified as fragments per kilobase of
transcript per million fragments mapped (FPKM) by dividing the total number of reads mapping to each
transcript by transcript length, and the total number of reads aligned to the genome divided by one
million. The Wilcoxon test from the statistical toolkit R was used to calculate differences in gene
expression between genes whose promoter contains an Auts2-marked site and all other genes. RNA-seq
data from this study are available in SRA (
SRR1298760 (replicate 2)).
6
Comprehensive RNA Extraction and Purification
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Total RNAs were prepared using the RNeasy maxi kit (Qiagen) and quality-controlled using RNA 6000 Nano assay (Agilent). Ribodepletion was performed using RiboMinus Transcriptome Isolation Kit (Human/Mouse; Thermo Fisher). mRNA purification was performed using Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA preps (Thermo Fisher). Absence of ribosomal RNA contaminations was controlled using RNA 6000 Pico assay (Agilent).
7
Biofilm-Grown S. aureus Transcriptome
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S. aureus MR23 was grown under biofilm conditions for 12 h in the presence or absence of 50 μM of the test compound. Cells were harvested by centrifugation at 5000×g at 25 °C for 10 min and immediately incubated with an appropriate volume of RNA Protect (Qiagen, Hilden, Germany) for 5 min. Cells were collected by centrifugation at 5000×g at 25 °C for 10 min and stored at −80 °C. Total RNA was extracted using an RNeasy Maxi Kit (Qiagen) according to the manufacturer’s instructions, except that cells were treated with 0.2 mg/mL lysostaphin at 37 °C for 30 min before extraction.
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S. aureus MR23 was grown under biofilm conditions for 12 h in the presence or absence of 50 μM of the test compound. Cells were harvested by centrifugation at 5000×g at 25 °C for 10 min and immediately incubated with an appropriate volume of RNA Protect (Qiagen, Hilden, Germany) for 5 min. Cells were collected by centrifugation at 5000×g at 25 °C for 10 min and stored at −80 °C. Total RNA was extracted using an RNeasy Maxi Kit (Qiagen) according to the manufacturer’s instructions, except that cells were treated with 0.2 mg/mL lysostaphin at 37 °C for 30 min before extraction.
8
RNA-Seq Library Preparation and Analysis
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mRNA extraction was performed by using RNeasy Maxi Kit (Qiagen). RNA-Seq libraries were prepared by using standard Illumina reagents and protocols. Paired-end sequencing with the read length of 50 bases were performed on the Illumina HiSeq 2000 platform following the manufacturer’s instructions. CASAVA 1.8.2 was used to demultiplex and to generate FASTQ files. The sequencing data were analyzed by using the software packages of Tophat mapping against hg19 and Cufflinks45 (link). The number of reads was normalized and expressed as fragments per kilobase of exon per million fragments mapped (FPKM).
9
Quantitative Analysis of Circular RNA and miRNA Targets
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Total RNA was isolated using TRIzol kit (Invitrogen) and purified in line with the instruction of the RNeasy Maxi kit (Qiagen, Dusseldorf, Germany). The cDNA synthesis was obtained using the PrimeScript RT Reagent Kit (Takara, Shiga, Japan). Then, qRT-PCR analysis was conducted utilizing the SYBR® Green PCR Master Mix kit (TaKaRa, Dalian, China). The level of NPRL3, circ_0037128, miR-497-5p and NFAT5 was quantified using the 2−∆∆Ct method, and U6 and β-actin were used for standardization [26 (link)]. The primers of the relative genes were listed as below: circ_0037128, F 5ʹ-CCAGTTCCCATCTCATGACC-3ʹ, R 5ʹ-GATGTGAAGCCGAACTACGCC-3ʹ; miR-497-5p, F 5ʹ-GCCGAGCAGCAGCACACTGTG-3ʹ, R 5ʹ-GTGCAGGGTCCGAGGT-3ʹ; NFAT5, F 5ʹ-CAGGCCAACCACAAAACGAG-3ʹ, R 5ʹ-TCATCTTGTGAGAAAGCCACA-3ʹ; NPRL3, F 5ʹ-ACGGCGATTCCAGGTTTTCA-3ʹ, R 5ʹ-GCATGCTGTAGCAGTGTTGG-3ʹ; β-actin, F 5ʹ-CTTCGCGGGCGACGAT-3ʹ, R 5ʹ-CCACATAGGAATCCTTCTGACC-3ʹ; U6, F 5ʹ-CTCGCTTCGGCAGCACATA-3ʹ, R 5ʹ-CGAATTTGCGTGTCATCCT-3ʹ.
10
Drought Tolerance in Rice Transcriptome
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Total RNA was extracted from the uppermost rice leaves, including the flag leaf of the plants of the two pairs of NILs, IR77298-14-1-2-B-10 (highly drought-tolerant), IR77298-14-1-2-B-13 (drought-susceptible), IR77298-5-6-B-18 (moderately drought-tolerant), and IR77298-5-6-B-11 (highly drought-susceptible), subjected to 1.0, 0.5 and 0.2 FTSW. Three replicates at the reproductive stage (a total of 45 independent RNA samples) were extracted using an RNeasy Maxi Kit (Qiagen, Valencia, CA). RNA concentration and quality were tested using a Nanodrop spectrophotometer (ND-1000; Nanodrop Technologies, Wilmington, DE) and a BioAnalyzer G2938A (Agilent Technologies, Santa Clara, CA).
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