As crude RNA extraction in PLRV was difficult due to its phloem limiting factor and the high concentration of polyphenols and polysaccharides in potato leaves and potato tuber, we tested earlier reported simple RNA isolation procedures to reduce the cost and make RPA easier [29 (link),34 (link)] to make a low-cost one-step RT-RPA for PLRV. This method was compared with an RNA isolation method based on the SpectrumTM Plant Total RNA kit (Sigma-Aldrich, St. Louis, MO, USA). We investigated several concentrations of sodium sulphite (0.25, 0.5, 0.75 and 1%) in simple RNA isolation from potato leaves and tubers to increase the quality of the RNA because the method itself was not very good at obtaining RNA from leaves and dormant tubers in the case of PLRV. To compare RT-PCR- and RT-RPA-based PLRV detection, purified RNA from the SpectrumTM Plant Total RNA kit (Sigma-Aldrich, St. Louis, MO, USA) was also employed. The RNA preparation technique utilizing cellular disc paper was used to detect PLRV in potato leaves in light of the findings.
Spectrum plant total rna kit
Manufactured by Merck Group
Sourced in United States, Germany, Italy, Sao Tome and Principe, United Kingdom, Poland, China, Spain, Australia, New Zealand, Norway, France, Canada, Switzerland, Macao, India
The Spectrum Plant Total RNA Kit is a lab equipment product designed for the isolation and purification of high-quality total RNA from a variety of plant tissues. The kit utilizes a column-based method to efficiently extract and purify RNA, making it suitable for downstream applications such as RT-PCR, Northern blotting, and microarray analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (47 mentions)
Dnase 1, by Thermo Fisher Scientific (47 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (39 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (33 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (33 mentions)
Iscript cdna synthesis kit, by Bio-Rad (28 mentions)
Lightcycler 480, by Roche (28 mentions)
2100 bioanalyzer, by Agilent Technologies (27 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (25 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (24 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (23 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (22 mentions)
Nanodrop, by Thermo Fisher Scientific (21 mentions)
Quantitect reverse transcription kit, by Qiagen (21 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (21 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (17 mentions)
Rq1 rnase free dnase, by Promega (15 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (15 mentions)
Hiseq 2000, by Illumina (14 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (14 mentions)
775 protocols using spectrum plant total rna kit
1
Simplified One-Step RT-RPA for PLRV Detection
2
Microalgae RNA Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Depending on cell density, 60–100ml of cultures was collected on 0.65 µm Durapore membrane filters, washed off the filter using 1ml of f/2 medium (N-deprived cells were washed with f/2 without nitrate supplement), and centrifuged at 13 000rpm for 1min at 4 °C. The supernatant was removed and pellets were flash frozen in liquid N2 and stored at –80 °C. Frozen samples were homogenized using a TissueLyser system (Qiagen) for 2×2min at 25 Hz. The samples were placed in a pre-cooled (–80 °C) adapter set for the first shaking step. Before the second shaking step, the samples were transferred to a room temperate adapter set, and 0.5ml of lysis buffer (SpectrumTM Plant Total RNA kit; Sigma-Aldrich) was added to each tube. Total RNA was isolated with a SpectrumTM Plant Total RNA kit (Sigma-Aldrich). To eliminate genomic DNA, an on-column digestion was performed using an RNAase-free DNase I set (Qiagen). Total RNA was quantified using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). The RNA quality was verified using formaldehyde gel electrophoresis. In addition, RNA integrity was checked on a 2100 Bioanalyzer (Agilent). All samples had RNA integrity numbers above 7.
3
Extraction and Quantification of Fungal RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Grapevine leaves with P. viticola lesions were cut in small fragments (only the lesioned area with fresh mycelia and sporangiophores), submerged in sterile water (20 ml) and the lower surfaces were gently brushed to remove the emerging fungal mycelia and spores. The water suspension containing the fungal matrix was centrifuged at 5,800 g for 10 min. The resulting pellet was washed with sterile water (1 ml) and centrifuged at 14,000 g for 1 min. The pellet was finally resuspended in lysis buffer (Spectrum Plant Total RNA kit from Sigma-Aldrich) and 0.5 ml of glass beads (0.1 mm) were added in the same tube (video protocol: youtube.com/watch? v = 9ZMrUGRzMvw&t=). Two cycles of beadbeater (FastPrep-24 MP Biomedicals) at maximum speed for 30 s alternated with 2 min pauses were used to disrupt the fungal tissues. After RNA extraction following the RNA kit instructions (Spectrum Plant Total RNA kit from Sigma-Aldrich), sample concentrations were measured using a spectrophotometer (Nanodrop 2000, Thermo Scientific). An arbitrary quantity threshold was set of 50 ng/µl and only 141 RNAs passed the check (74 from Italy and 67 from Spain). Selected samples were then pooled to have the same final concentration (7 ng/µl; Supplementary Table S1 ), resulting in seven pools from Italy and nine pools from Spain.
4
Efficient Total RNA Extraction from Mycelia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For total RNA extraction, 100 mg of mycelia were resuspended in lysis buffer (Spectrum Plant Total RNA kit; Sigma-Aldrich); in the same tube, 0.5 ml of glass beads (0.1 mm) was added, and the samples were mixed in a beat beater (Qiagen TissueLyser II) at maximum speed for 20 to 30 s and immediately placed in ice. After total RNA extraction according to the Spectrum Plant Total RNA kit instructions (Sigma-Aldrich), the samples were measured using a UV spectrophotometer to determine the concentration, and electrophoresis in agarose gels indicated the quality. Only samples with a minimum of 40 ng/ml were included in pools for high-throughput sequencing (i.e., the 248 samples described above). The high-quality samples were combined in each pool, resulting in 17 pools from Spain and 12 pools from Italy.
5
Quantitative Transcriptomic Analysis of Arabidopsis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For qRT-PCR analyses, total RNA was extracted using the Spectrum Plant Total RNA kit (Sigma-Aldrich) according to the manufacturer's instructions. cDNAs were prepared from 2 mg of total RNA using M-MLV reverse transcriptase (Promega) and oligo-dT 18 . Gene expression levels were determined by qPCR with SYBR Green I Mastermix (Enzynomics) and 0.5 mM of the forward and reverse primers for each gene. Relative expression levels of each gene were normalized to PP2A mRNA levels. Gene-specific primers used for qPCR are listed in Supplemental Dataset 5.
For the microarray analyses, biological triplicate samples were taken from among 2.5-day-old dark-grown seedlings of the pifQ and SCR:PIF1/pifQ lines. Total RNA was extracted from these samples using the Spectrum Plant Total RNA kit (Sigma-Aldrich). The Agilent Arabidopsis Genome 44 k chip was used for the microarray analysis. Microarray data were analyzed using R with packages from Bioconductor. The limma package was used for background correction and for normalization within arrays and between arrays (Smyth, 2005) . Then, a linear model was fitted using lmFit followed by statistical calculations using ebayes. Genes having more than 2-fold changes and FDR values of less than 0.05 were identified as DEGs.
For the microarray analyses, biological triplicate samples were taken from among 2.5-day-old dark-grown seedlings of the pifQ and SCR:PIF1/pifQ lines. Total RNA was extracted from these samples using the Spectrum Plant Total RNA kit (Sigma-Aldrich). The Agilent Arabidopsis Genome 44 k chip was used for the microarray analysis. Microarray data were analyzed using R with packages from Bioconductor. The limma package was used for background correction and for normalization within arrays and between arrays (Smyth, 2005) . Then, a linear model was fitted using lmFit followed by statistical calculations using ebayes. Genes having more than 2-fold changes and FDR values of less than 0.05 were identified as DEGs.
6
RNA-Seq Workflow for Plant Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For each sample, liquid nitrogen frozen tissue sample (100 mg) was ground using a mortar and pestle cooled in presence of liquid nitrogen. A SpectrumTM Plant Total RNA kit (Sigma-Aldrich, St. Louis, MO, United States) was employed according to the manufacturer’s instructions. Quality control was performed on a Thermo Scientific NanoDrop with all 260/280 nm and 260/230 nm readings above 1.8. Illumina TruSeq RNA libraries were constructed per the manufacturer’s protocol, with quality evaluated using the Agilent Bioanalyzer. Samples were sequenced on a HiSeq 2000 instrument (Illumina, San Diego, CA, United States). Six lanes were multiplexed with five samples per lane in the single ended, 50 bp configuration. A mean of 41 M reads passing quality filters was obtained for each sample (min. 31 M, max. 50 M). Cluster generation used the cBot SR Cluster Gen Kit (V3) and the flow cell used was SR FC Information (V3).
7
RNA Extraction from Frozen Leaves
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen leaves were crushed using two glass beads in a FastPrep-24TM 5G homogenizer (MP Biomedicals) for 6 s at speed 5.0. RNA extraction was done using the SpectrumTM Plant Total RNA Kit (Sigma Aldrich) as described by the manufacturer. Potential residual DNA was removed by addition of RNase free DNase from Qiagen along with RNasin® Ribonuclease inhibitor (Promega). Subsequently, the samples were purified using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel) with the NTC buffer. RNA concentration and quality were evaluated by NanoDrop 1000 (ThermoFisher Scientific) and on a 1% agarose gel to visualize the 18S and 28S ribosomal bands.
8
RNA Extraction and Quantification from Phloem
9
Strawberry Rhizome Total RNA Extraction and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from strawberry rhizomes using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. In addition, 30 min on-column DNase digestion was performed on the isolated RNA to remove traces of DNA contamination (Sigma-Aldrich, USA). The quantity and quality of the isolated RNA was assessed using the Nanodrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA), and the Agilent RNA 6000 Nano kit in the Agilent 2100 Bioanalyzer (Agilent Technology, Santa Clara, CA, USA), respectively. The RNA integrity numbers (RIN) for the isolated RNA samples ranged from 6.8 to 9.5 among the 36 samples that were used for library preparation and sequencing. The libraries were prepared using the TruSeqTM stranded total RNA library prep kit (Illumina), and the samples were indexed and sequenced using four lanes on an Illumina HiSeq 3/4000 (2 × 150 bp) System by the Norwegian Sequencing Centre, Oslo, Norway.
10
RNA Extraction from Frozen Mycelium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen mycelium was used for RNA extraction with the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich). RNA concentration (µg/mL) and purity (A260/A280 ratio) were determined using a 1.5-μL aliquot on a NanoDropTM spectrophotometer (Thermo Fisher Scientific, Madrid, Spain). Samples were diluted to 0.1 μg/μL and treated with DNAse I (Thermo Fisher Scientific) to remove genomic DNA traces that could be co-extracted with RNA.
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!