All strains were grown in GMM + YE at 37°C/250 rpm for 18 hr. After incubation, extraction of total RNA was performed using the Qiagen RNEasy Mini Kit, following manufacturer's recommendations. Six micrograms of total RNA from each sample were digested with RNase‐free Turbo DNase (Invitrogen, Carlsbad, California) following the manufacturer's protocol. Next, cDNA was synthesised using the SuperScript II system (Invitrogen), according to manufacturer's instructions. Quantitative reverse transcription PCR was performed using SYBR® Green Master Mix (Bio‐Rad, Hercules, California) in a CFX Connect Real‐Time System (Bio‐Rad). RasA and β‐tubulin (TubA) specific qPCR primers were designed to flank introns, where possible. Transcript levels were calculated by comparative ΔCt and normalised to β‐tubulin. Each experiment was performed in biological triplicate.
Rnase free turbo dnase
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania
RNase-free TURBO DNase is a high-performance DNA-specific endonuclease that efficiently degrades DNA in the presence of RNA. It is an essential tool for removing contaminating DNA from RNA samples, ensuring the integrity of downstream RNA-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Rneasy mini kit, by Qiagen (6 mentions)
Superscript 2 system, by Thermo Fisher Scientific (2 mentions)
Cfx connect real time system, by Bio-Rad (2 mentions)
Sybr green master mix, by Bio-Rad (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Sybr green universal pcr master mix protocol, by Thermo Fisher Scientific (2 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Genelute total rna purification kit, by Merck Group (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Lightcycler 480 sybr green 1 master, by Roche (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Nebnext ultra 2 directional rna library prep kit, by Illumina (1 mentions)
Scriptseq v2 rna seq kit, by Illumina (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
24 protocols using rnase free turbo dnase
1
Quantitative Gene Expression Analysis
2
Quantitative RT-PCR for Gene Expression
3
Quantifying Gene Expression in Arabidopsis
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Seedlings were grown on 1/2 MS medium vertically under constant-dark conditions for 2 days. The apical hooks were harvested and frozen in liquid nitrogen and stored at −80°C. Total RNA was purified using the RNeasy Plant Mini kit (QIAGEN) or the GenElute Total RNA Purification Kit (Sigma-Aldrich) in accordance with the manufacturer’s recommendations. Genomic DNA was removed using RNase-free Turbo DNase (Invitrogen), and single-stranded cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad) with 1 μg of RNA. Q-PCR was performed with the Light Cycler 480 SYBR Green I Master (Roche) in the 96-well CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Expression data were normalized against ubiquitin 10 using the 2−ΔΔCT method (37 (link)). Three biological replicates were performed for each line, and two to three technical replicates were performed for each biological replicate. Primers used in this study are listed in table S1.
4
Isolation and Analysis of dsRNA
5
Isolation and Transfection of Mitochondrial RNA
6
RNA Extraction and Sequencing Workflow
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Total RNA was prepared using the mirVana miRNA Isolation Kit (Life Technologies) or RNeasy Plus Mini kit (Qiagen) and treated with RNase-Free Turbo DNase (Ambion) or RNase-free DNase Set (Qiagen) according to manufacturers’ instructions. RNA integrity was assessed using RNA Reagents and RNA Screentapes on a Tapestation as part of quality control. PolyA + RNA libraries were then prepared using the ScriptSeq v2 RNA-seq Kit (Epicentre, Madison, WI, USA) or the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina. All RNA-seq experiments were performed in triplicate in accordance with ENCODE consortium guidelines (The_ENCODE_Consortium, 2016 ).
7
In situ hybridization of adult gonads
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In situ hybridization on whole mount sample and sections were performed as described 27 (link), 32 (link) with some minor modifications. Adult gonads were dissected and cut into small pieces, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4) (4% PFA-PBS) at 4°C overnight, followed by buffer changes with 0.5 M sucrose in PBS at 4°C overnight. The samples were embedded in Optimal Cutting Temperature and cryosectioned at 8 μm for ovaries and 5 μm for testes on the Leica RM2135 Microtomes (Leica, Germany). The cryosections were mounted on frost glass slides (Fishery, USA) and stored at -80°C before use. pLcvasa854 containing the 854-bp Lcvasa cDNA fragment was linearized with Apa I and Sac II for the synthesis of sense and anti-sense riboprobes from Sp6 or T7 promoter by using the digoxigenin (DIG) or FITC RNA Labeling Kit (Roche). The probes were treated with RNase-free TURBO DNase (Ambion) and purified. Chromogenic and fluorescent in situ hybridization (FISH) was carried as described 32 (link). After extensive washes in PBS in darkness, the slides were stained for nuclei with DAPI (1 µg/ml) for 10 min at room temperature, washed three times in PBS and mounted for microscopy.
8
Transcriptome Analysis of S. scabiei
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Total RNA was isolated from the five replicate samples of each S. scabiei Ee, Le and Af (≤2 h after isolation from skin crusts) using the TRIzol™ reagent (cat. no. 15596026, Thermo Fischer Scientific Inc., Waltham, MA, USA) and treated with RNase-free TURBO DNase (Ambion®, cat no. AM1907, Thermo Fisher Scientific). Libraries for sequencing were constructed from mRNAs and small RNAs from each Ee, Le and Af using TruSeq Stranded Total RNA Library Prep Kit (Illumina Inc., San Diego, CA, USA) and TruSeq Small RNA Library Prep Kit (Illumina), respectively. In brief, mRNA was isolated using magnetic beads with Oligo (dT), cleaved into short fragments, transcribed into complementary DNA (cDNA) via reverse synthesis, connected with adaptors and amplified using PCR for mRNA-enriched libraries. For small RNA libraries, RNAs of 20–30 nucleotides (nt) in length were selected using electrophoresis, ligated to adaptors and amplified. All libraries were evaluated using the Agilent 2100 Bioanalyzer and ABI StepOnePlus real-time PCR system and then sequenced using the Illumina HiSeq™ 4000 platform.
9
Quantitative RNA Expression Analysis
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RNA from 300 Bam>>v105115 testes or v105115 undriven testes was extracted using TRIzol (Invitrogen). Extracted total RNA was treated with RNase-free Turbo DNase (Ambion) and purified using an RNeasy mini kit (Qiagen). 1 μg of DNAse treated and purified RNA was used for cDNA synthesis with the Transcriptor First Strand cDNA Synthesis kit (Roche) according to the manual in a 20 μl reaction. qPCR reactions were set up with 10 μl iTaq™ Universal SYBR® Green Supermix (Bio-Rad), 4 μl ddH2O, 0.5 μl (10 μM) gene-specific primer A, 0.5 μl (10 μM) gene-specific primer B and 5 μl cDNA (1:25 dilution). qPCR was performed in three technical replicates on the Mx3000P qPCR. Ct values were normalized to the mRNA expression level of Rpl32. Mean value and SEM of three biological replicates was calculated using GraphPad PRISM version 5.03. For statistical analysis one-sample t-test with a hypothetical value of 1 was used.
10
Diurnal Transcriptional Profiling of Seedlings
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About 20 seedlings of 12–14 days after stratification (das) were collected every 4 h over a diurnal or circadian cycle. RNA was purified using the Maxwell 16 LEV simply RNA Tissue kit (Promega). RNA was incubated with RNase-free TURBO DNase (Ambion) to eliminate genomic DNA contamination. Single strand cDNA was synthesized using 1 μg of RNA using iScriptTM Reverse Transcription Supermix for RT-Q-PCR (Bio-rad) or AffinityScript Q-PCR cDNA Synthesis Kit (Agilent). For qPCR analysis, cDNAs were diluted fivefold with nuclease-free water and qPCR was performed with 10% of diluted cDNA with Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent) or iTag Universal SYBR Green Supermix (Bio-Rad) in a 96-well CFX96 Touch Real-Time PCR detection system (Bio-Rad). The ISOPENTENYL PYROPHOSPHATE:DIMETHYLALLYL PYROPHOSPHATE ISOMERASE 2 (IPP2) gene was used as control (Huang et al., 2012 (link)). Data were analyzed using the second derivative maximum method. Resulting Cp values were converted into relative expression values using the comparative Ct method (Livak and Schmittgen, 2001 (link)).
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