Total RNA was extracted from tissues using the Plant RNA mini spin kit (MACHEREY-NAGEL GmbH and Co. KG, Neumann-Neander-Straße 6-8, Duren, Germany) with the in-column DNase I treatment according to the manufacturer's instructions. Sample quantity and purity were determined using a NanoVue Plus Spectrophotometer (GE Healthcare). RNA samples with an OD260/OD280 absorbance ratio between 1.9 and 2.2 were used for further analysis, and RNA integrity was assessed on 1% denaturing formaldehyde agarose gel. For each sample, 1 μg of total RNA was reverse transcribed using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) and oligo (dT) primers according to manufacturer's instructions. The reverse transcribed cDNAs were then diluted 1:12 with nuclease-free water and used for qPCR analysis.
Plant rna mini spin kit
Manufactured by Macherey-Nagel
Sourced in Germany
The Plant RNA mini spin kit is a laboratory equipment designed for the rapid and efficient extraction of high-quality total RNA from various plant tissues. The kit utilizes a spin column-based method to isolate RNA, ensuring a streamlined and reliable purification process.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 4 fluorometer, by Thermo Fisher Scientific (2 mentions)
Superscript first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Nanovue plus spectrophotometer, by GE Healthcare (1 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
4 protocols using plant rna mini spin kit
1
Total RNA Extraction and cDNA Synthesis
2
Transcriptome Analysis of Capsicum chinense Cultivars
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Green fruits (16 dpa) from six different cultivars of C. chinense were used for whole-transcriptome sequencing. Total RNA was isolated from the pooled tissues of three biological replicates for each cultivar by using the plant RNA mini spin kit (Macherey-Nagel). Total mRNA was isolated, fragmented, and reverse-transcribed into cDNA. Double-stranded cDNA was then purified by using 1.8x Agencourt AMP XP beads. Sequencing libraries were constructed by using the NEBNext Ultra II RNA Library Prep Kit according to the manufacturer’s protocol. To confirm accuracy before sequencing, the insert size and integrity of the libraries were analyzed with an Agilent 2100 Bioanalyzer (Invitrogen), and the Qubit 4 Fluorometer (Invitrogen) was used for library quantification. The RNA sequencing library from each sample was sequenced in the Illumina NextSeq. 500 platform to produce paired-end reads.
3
Pepper ABC Transporter Gene Expression
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Total RNA was isolated from pepper fruits (6, 16 and 25 dpa) by using the Plant RNA mini spin kit (Macherey-Nagel). First-strand cDNA was synthesized with 1 μg total RNA per sample by using the Super Script First-Strand Synthesis system (Invitrogen). To identify in the three Capsicum genomes the orthologs of the markers previously reported by [5 (link)] for the ABC transporter family, the CDS sequences for the CA06g14430 and CA11g09150 genes were downloaded from the Sol Genomics database (https://solgenomics.net/ ) [49 (link)] and a BLASTN search was performed (identity ≥ 98% and coverage ≥ 70%) across the three pepper genomes. Gene-specific primers for the selected Capsicum ABC transporter orthologs were designed by using Primer3Plus (http://www.primer3plus.com/ ). The qRT-PCR analysis involved a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with a total volume of 20 μL containing 1 μL cDNA template, 2 μL forward and reverse primers (10 μM), 10 μL SYBR Green PCR Master (ROX) (Roche, Shanghai) and 7 μL sterile distilled water. For each sample, three replicates were run to compute the average Ct values. The data were analyzed by the 2−ΔΔCt method [50 (link)]. Relative gene expression was normalized against that of the endogenous control β-tubulin [51 (link)].
4
Transcriptome Analysis of Capsicum chinense
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Green fruits (16 dpa) from two different cultivars of C. chinense were used for whole-transcriptome sequencing. Total RNA was isolated from the pooled tissues of three biological replicates for each cultivar with the Plant RNA mini spin kit (Macherey-Nagel). The quantity and quality of the total RNA were analyzed with the Agilent 2100 Bioanalyzer and Qubit 4 Fluorometer (Invitrogen), respectively. The RNA sequencing libraries were prepared by using the NEBNext Ultra II RNA Library Prep Kit according to the manufacturer's protocol. The mRNAs were enriched by using magnetic beads with Oligo (dT), then fragmented into shorter fragments with a fragmentation buffer. The first-strand cDNA was synthesized from the fragmented mRNA with a random hexamer primer. The resulting cDNAs were added to sequencing adapters, and sequencing primers were used for library amplification. The insert size of the library was analyzed with Agilent 2100 Bioanalyzer (Invitrogen), and the Qubit 4 Fluorometer (Invitrogen) was used for library quantification. The RNA sequencing library from each sample was sequenced in the Illumina NextSeq 500 platform with paired-end sequencing. The resulting image files were converted to FASTQ with 2x75-bp reads. The Illumina reads were deposited with the Sequence Reads Archive (NCBI) under the following accession number PRJNA526219.
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