RNA was extracted from nectaries by mechanical disruption with a microcentrifuge pestle, and using the RNAqueous®-Micro RNA isolation kit (Ambion, Austin, TX) with Plant RNA Isolation Aid (Ambion, Austin, TX). Agarose gel electrophoresis and UV spectrophotometry were used to assess RNA quality of all samples prior to submission to the University of Minnesota Genomics Center for barcoded library creation and Illumina HiSeq 2500 sequencing. Six TruSeq RNA v2 libraries were created from ~500 ng of total RNA (triplicate samples for immature and mature nectaries) and sequenced via 50 bp, paired-end runs on the HiSeq 2500 using Rapid chemistry. All libraries were pooled and sequenced across two lanes to achieve the equivalent of one lane output. This generated ~368 M total reads (~184 M paired-end reads) and the average quality scores were above Q30.
Plant rna isolation aid
Manufactured by Thermo Fisher Scientific
Sourced in United States
Plant RNA Isolation Aid is a reagent designed to facilitate the extraction and purification of high-quality RNA from plant tissues. It is intended to assist in the isolation process, providing a streamlined approach for obtaining RNA samples suitable for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Mirvana mirna isolation kit, by Thermo Fisher Scientific (6 mentions)
Rnaqueous rna isolation kit, by Thermo Fisher Scientific (5 mentions)
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Rnaqueous kit, by Thermo Fisher Scientific (3 mentions)
Moloney murine leukemia virus reverse transcriptase, by Promega (3 mentions)
Nanodrop, by Thermo Fisher Scientific (3 mentions)
Truseq rna sample preparation kit, by Illumina (2 mentions)
Thermal cycler tp800, by Takara Bio (2 mentions)
Nucleospin rna virus kit, by Macherey-Nagel (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Quantstudiotm 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Nucleospin rna plant isolation kit, by Macherey-Nagel (2 mentions)
Rnaqueous micro rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500 sequencing, by Illumina (1 mentions)
Mighty ta cloning reagent set for primestar, by Takara Bio (1 mentions)
Superscript first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Primestar gxl dna polymerase, by Takara Bio (1 mentions)
38 protocols using plant rna isolation aid
1
Transcriptome Analysis of Nectary Development
2
Cloning and Sequencing of Carotenoid Biosynthesis Genes
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Total RNA was isolated from the algal cells with RNAqueous kit (Ambion) and Plant RNA Isolation Aid (Ambion). First-strand cDNA was synthesized with SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) from total RNA treated with DNase I (Invitrogen). cDNAs containing EgcrtE and EgcrtB coding sequences were amplified by RT-PCR with PrimeSTAR GXL DNA Polymerase (Takara Bio). Primers used for RT-PCR were as follows: EgcrtE, 5′-TTTCGCTCACACGCACAATG-3′ and 5′-CCCAGCGTACAGAAAAGCTA-3′; EgcrtB, 5′-TTCGGTCGCTCCCCTTCCA-3′ and 5′-AGCAGCCGAGTATGATACGA-3′. The amplified fragments were gel-purified (Gel/PCR Extraction kit, FastGene) and sub-cloned into pMD20-T vector with Mighty TA-cloning Reagent Set for PrimeSTAR (Takara Bio) and sequenced. E. coli strain JM109 (Takara Bio) was used as a host for the plasmids and grown in LB medium [54 (link)] at 37 °C in the dark. Ampicillin (50 μg ml−1) was added to the medium as needed.
3
Isolation and RNA Extraction from Plant Ovules
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Total RNA for RNA sequencing (RNA-Seq) and quantitative reverse transcription polymerase chain reaction was extracted from ovules surgically isolated from pistils, which were pollinated with WT and gcs1/gcs1 pollen or were without pollination, using a pin and a needle. Only ~20 ovules at the upper region (around the top one-third of pistil) were sampled from a pistil. Isolated ovules were pooled into a droplet of 5 μl of RNAlater on the tip of the pestle. After the collection of 150 to 200 ovules within 30 to 60 min, excess RNAlater was removed. The pestle was inserted into a precooled 1.5-ml tube, and tissues were ground with liquid nitrogen. Two hundred microliters of lysis buffer for the RNAqueous-Micro Total RNA Isolation Kit (Life Technologies) and 175 μl of lysis buffer + 25 μl of Plant RNA Isolation Aid (Ambion) were added into the tube, and the samples were stored at −80°C until subsequent RNA extraction procedures. Total RNA extraction was performed according to the manufacturer’s instructions, where total RNA was eluted in 24 μl of elution solution. The RNA was quantified using the Quant-iT RiboGreen RNA Assay Kit (Life Technologies) with the EnSpire Multimode Plate Reader (PerkinElmer) and qualified by the RNA integrity number analysis using the Agilent RNA 6000 Nano Kit (Agilent Technologies).
4
Transcriptome Analysis of Capitulum Tissues
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Disc florets and bracts were scraped off each capitulum and grinded in liquid nitrogen. Total RNA of 100 mg grinded material was extracted using the RNAqueous Kit with addition of the Plant RNA Isolation Aid (Ambion, Applied Biosystems, USA). Samples were treated with DNase I (Invitrogen, USA) for 20 min to remove remaining DNA. Sample purity, integrity and concentration were assessed using the RNA 6000 Nano Reagent kit in a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California, USA).
Sequencing libraries were prepared using an Illumina TruSeq RNA Sample Preparation Kit following the manufacturer’s instructions. Libraries were sequenced using the Illumina HiSeq 2000 system at the Centre Nacional d'Anàlisi Genòmica (CNAG:https://www.cnag.crg.eu/ , Barcelona, Spain) as unstranded single-end reads of 50 bp in length. The original data set was deposited at the NCBI Sequence Read Archive (Submission ID: SUB5575431, BioProject ID: PRJNA561716).
Sequencing libraries were prepared using an Illumina TruSeq RNA Sample Preparation Kit following the manufacturer’s instructions. Libraries were sequenced using the Illumina HiSeq 2000 system at the Centre Nacional d'Anàlisi Genòmica (CNAG:
5
RNA Isolation and Sequencing for Germination and TF Mutant Analysis
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For the samples collected from the time course of germination (Col-0 only; Fig. 1a (i)), the Ambion Plant RNA isolation aid and RNAqueous RNA isolation kit were used for RNA isolation. RNA quality and integrity were determined using the Nanodrop 1000 Spectrophotometer and Agilent Bioanalyser. Only high-quality RNA samples (Abs260/280 nm ratios of 2.0–2.1) were used for RNA-seq library generation with the Illumina TruSeq Total RNA sample prep kit. RNA-seq libraries were multiplexed and loaded per lane into the Illumina HiSeq flow cell v3. All sequencing protocols were carried out as per the manufacter’s instructions using the Illumina HiSeq 1000 and HiSeq control software.
For the samples collected from the eight TF mutant lines and Col-0 in parallel (validation of the DREM predictions), RNA from the 24 h SL timepoint was extracted with the Spectrum RNA extraction kit (Sigma) in duplicates for each genotype. RNA-seq libraries were prepared with the Illumina TruSeq mRNA kit, pooled and sequenced on one NextSeq500 flow cell.
For the samples collected from the eight TF mutant lines and Col-0 in parallel (validation of the DREM predictions), RNA from the 24 h SL timepoint was extracted with the Spectrum RNA extraction kit (Sigma) in duplicates for each genotype. RNA-seq libraries were prepared with the Illumina TruSeq mRNA kit, pooled and sequenced on one NextSeq500 flow cell.
6
Total RNA Extraction from Plant Tissues
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The apical buds from NL and ANL branches were mixed in equal amount separately to extract the total RNAs using Total RNA Purification Kit (LC sciences, #TRK-1001) following the manufacturer’s instructions. Briefly, 100 mg of tissues was grinded into powder in liquid nitrogen; then, 600 µl of extraction buffer with 6% Plant RNA Isolation Aid (Ambion, #Am9690) was added. The mixture was shaken vigorously, then incubated on ice for 15 min and centrifuged at 12,000 rpm for 10 min at room temperate, by which the yield of total RNA could be improved.
7
Leaf RNA Extraction and Purification
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After three months of continuous treatment, healthy leaves from three individual plants in each chamber were harvested after physiological measurements. Samples were immediately frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted and purified using RNAqueous phenol-free total RNA isolation (Ambion, Austin, TX, USA) and Plant RNA Isolation Aid (Ambion) following the manufacturer's instructions and checked for RNA integrity number to assess the RNA integration with an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, USA).
8
High-Quality Total RNA Isolation
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Frozen leaf tissues were macerated in the presence of liquid N2 in an RNase free, pre-chilled mortar and pestle. Tissue was processed in ~200 mg portions using the RNAqueous™ Midi large scale phenol-free total RNA isolation kit (Ambion) according to the manufacturer’s instructions with the following exceptions: 1) lysis buffer solution was made fresh for each isolation, 2) Plant RNA Isolation Aid (Ambion) was added to each lysis buffer prep in a 1:8 ratio by volume. Total RNA was assessed on the Agilent Bioanalyzer using the RNA 6000 Nano kit (Agilent) with the Plant Total RNA assay. High quality total RNA samples (28s/18s ratio ≥1.7; RIN ≥8; A260/A280: ≥1.8) from individual biological replicates were pooled. Biological replicates were never mixed except prior to normalized library construction.
9
RNA Isolation and RT-qPCR Analysis for Gene Expression
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RNA was isolated using the InviTrap Spin plant RNA mini kit (Invitek Molecular, Washington, DC, USA; # 1064100300) with the addition of 25 µL Plant RNA isolation Aid (Ambion, Austin, TX, USA), followed by a DNAse treatment (TURBO DNA-free kit; Invitrogen, Waltham, MA, USA). cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. Quantitative real-time RT-PCR (RT-qPCR) was performed using a BioRad MyiQ PCR machine with the SYBR green mix as described in Horstman et al. (2015) (link). Relative gene expression was calculated with the 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)) using the nonDEX treated (mock) samples as calibrators and the SAND gene (Czechowski et al., 2005 (link)) as the reference. Three biological replicates comprising germinating seeds/seedlings were used for each treatment. Statistically significant changes in gene expression levels were determined using Student’s t test P < 0.05. The qPCR DNA primers are shown in Supplemental Table S3 .
10
RNA-seq of Fungal Strains under Stress
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RNA-seq was performed for strains F-3808 and F-4515 grown in control conditions (malt agar, temperature 25 C) and under low temperature and high salinity (). Prior to RNA extraction, samples were collected in RNAlater solution (Ambion, USA), then homogenized using liquid nitrogen. Extraction was carried out using RNeasy Mini Kit (Qiagen, Germany) following manufacturer’s instruction. The only modification was the addition of 10% Plant RNA Isolation Aid (Ambion, USA) to the lysis buffer. RNA quality was assessed using capillary electrophoresis on Bioanalyzer 2100 (Agilent, USA), only RNA with integrity number (RIN, [46 (link)]) greater or equal to 8 were taken for library preparation. For library preparation, TruSeq RNA Sample Prep Kit v2 (Illumina, USA) was used following manufacturer’s instructions. After preparation libraries were quantified using Qubit fluorometer and quantitative PCR and sequenced on HiSeq2000 with read length 51 nucleotide.
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