Total RNA was isolated from leaves of individual young seedlings with TRI Reagent RT (MRC Inc., United States) and treated with RQRNaseFree DNase (Promega Corporation, Madison, WI, USA) according to manufacturers’ recommendations. RNA was reverse-transcribed to cDNA with a RevertAid H Minus First Strand cDNA Synthesis Kit (Thermoscientific). Amplification primers specific to the N-terminal tail of the αCENH3 gene from rye, wheat (Genbank accession nos. MG384772.1, JF969285.1) and triticale cDNA had been designed in (Evtushenko et al. 2017 (link)). The amplification products were cloned by using an InsTAclone PCR Cloning Kit (Thermoscientific). Both strands of 15–20 clones from each parental variety and TDK lines were sequenced using BigDye Terminator Cycle Sequencing chemistry (v. 3.1) on an ABI3100 Genetic Analyzer (Applied Biosystems, CA, USA). Coding sequences of CENH3 were aligned with online Clustal Omega (Sievers et al. 2011 (link)) at http://www.ebi.ac.uk/Tools/msa/clustalo .
Rq rnase free dnase
Manufactured by Promega
Sourced in United States
RQ-RNase-free DNase is a lab equipment product designed for the removal of DNA contamination from RNA samples. It is an RNase-free DNase that effectively degrades DNA without affecting the integrity of the RNA.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
Goscript reverse transcription kit, by Promega (4 mentions)
Trizol reagent, by Takara Bio (4 mentions)
Sybr green, by Takara Bio (3 mentions)
Cfx96 real time pcr system, by Bio-Rad (3 mentions)
Eco 3, by Illumina (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Instaclone pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
Abi 3100 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Oligo dt primer, by Takara Bio (1 mentions)
Sybr green master mix, by Roche (1 mentions)
Rnase h, by Toyobo (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Cfx manager software, by Bio-Rad (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Rnaiso reagent, by Takara Bio (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Random hexamer primer, by Thermo Fisher Scientific (1 mentions)
13 protocols using rq rnase free dnase
1
CENH3 Sequence Characterization in Rye, Wheat, and Triticale
2
Quantitative Analysis of Rice Gene Expression
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Total RNA was isolated from the one-month-old rice leaves using the TRIzol reagent (Takara, Dalian, China), and the genomic DNA was removed by treatment with RQ-RNase free DNase (Promega, Madison, WI, USA). Complementary DNA was synthesized using the GoScript Reverse Transcription Kit (Promega) following the manufacturer’s instructions. A BIO-RAD CFX96 Real-time PCR system (Bio-Rad, Hercules, CA, USA) and SYBR-Green (Takara) were used for the qRT-PCR analyses. The gene expression levels were normalized to that of the level of Ubiquitin. The primers used for qRT-PCR are listed in Additional file 4 : Table S1.
3
Total RNA Isolation and qRT-PCR Analysis
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Cellular total RNA was isolated by using the TRIzol reagent (Takara, Dalian, LN, China), and the RNA was treated with RQ-RNase-free DNase (Promega, Madison, WI, United States) to eliminate genomic DNA contamination. The GoScript Reverse Transcription kit (Promega, Madison, WI, United States) was used to synthesize cDNA. Quantitative RT-PCR was performed using the Illumina Research Quantity software Illumina Eco 3.0 (Illumina, San Diego, CA, United States), and each gene expression was normalized against that of the Ubiquitin level. The primers used for qRT-PCR are listed in Table 1 .
4
Quantitative RT-PCR Analysis of Gene Expression
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Total cellular RNA was extracted using TRIzol (Life Technologies, USA) and treated with DNase using RQ RNase-free DNase (Promega, China) prior to cDNA production. The cDNA was reverse-transcribed from 1 μg of total RNA using oligo(dT) primers according to the manufacturer's protocol (TaKaRa, China). Quantitative RT-PCR was carried out using SYBR Green Master Mix (Roche) and specific primer sets (Table 1 ). Amplification reactions were performed under the following conditions: 2 min at 50°C, 10 min at 95°C, 40 cycles for 15 s at 95°C, and 1 min at 60°C. Relative transcript levels were calculated using the ΔΔCt method as specified by the manufacturer.
5
Quantitative RT-PCR Gene Expression Analysis
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Total cellular RNA was isolated using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. RNA concentration was measured using a NanoDrop ND-1000 spectrophotometer. The samples were treated with RQ-RNase-free DNase (Promega, Madison, WI, USA). An RNaseH (Toyobo, https://www.toyobo-global.com/ ) reverse transcription kit was used to synthesize cDNA, according to the manufacturer’s instructions (Promega). qRT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Amplification and quantification were performed with gene-specific primers using CFX Manager software (Bio-Rad), and values were normalized against internal UBIQ1. Three biological and two technical replicates were used for each analysis [46 (link)]. All the primers used for qRT-PCR are listed in Table S1 .
6
Quantifying EBV Gene Expression in Tumor Tissue
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Snap‐frozen tumor tissues were sliced in 5 × 10 mm sections and homogenized in 1 mL Trizol (Life Technologies, Waltham, MA). RNA was treated with RQ RNase‐free DNase (Promega, Madison, WI) followed by target‐specific cDNA synthesis as described in detail recently.25 cDNA was diluted 10 times or higher for use in SYBR Green based Real‐Time PCR quantification of each target gene (LightCycler480, Roche, Almere, the Netherlands). Quantification was calculated via a dilution curve of a plasmid pool containing all target genes and specificity was confirmed by melting curve analysis. Cellular housekeeping gene (U1A) was used as RNA quality control and for normalizing transcript levels.25 Correction for remaining viral DNA in the DNase‐I treated RNA extracts was done for all non‐spliced targets (Rta, PK, TK, VCA‐p18) to exclude genomic EBV DNA contamination. For this the viral load PCR was used targeting a genomic region not included in the cDNA target sequences.24
7
Isolation and Analysis of Centromeric Histone
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Total RNA was isolated from leaves of 12dayold seedlings using the TRI Reagent (MRC, Inc., USA) and treated by RQRNaseFree DNase (Promega, Madison, WI) according to the manufacturer’s instructions. RNA was reverse-transcribed to cDNA using a RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The specific primers used to amplify the CENH3 gene from cDNA were:
1) 5’ATGGCCCGCACCAAGCAC3’, 5’GCATCACCAAAGCCTCC3’, to amplify the coding region of αCENH3; and
2) 5’TGGGTCGCACGAAGCAC3’, 5’TCACCAAAGCCTTCTCCCC3’, to amplify the coding region of βCENH3.
1) 5’ATGGCCCGCACCAAGCAC3’, 5’GCATCACCAAAGCCTCC3’, to amplify the coding region of αCENH3; and
2) 5’TGGGTCGCACGAAGCAC3’, 5’TCACCAAAGCCTTCTCCCC3’, to amplify the coding region of βCENH3.
8
Total RNA Isolation and RT-qPCR Analysis
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Total RNA was isolated using the RNeasy Plant Mini Kit (QIAGEN, Duesseldorf, Germany) and treated with RQ-RNase free DNase (Promega, Madison, WI, United States) to remove contaminating genomic DNA. For cDNA synthesis, reverse transcriptase minus RNase H (Toyobo)3 was used, according to the manufacturer’s instructions. The products obtained in RealTime Quantitative PCR (RT-qPCR) were quantified using Illumina Research Quantity software, Illumina Eco 3.0 (Illumina, San Diego, CA, United States) and the values were normalized against the tubulin levels in the same samples. The primers used for qRT-PCR are listed in Supplementary Table 1 .
9
Hypothalamus and Pituitary RNA Extraction
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The total amount of total RNA from the hypothalamus and pituitary samples was extracted using RNAiso Reagent (TaKaRa Bio, Kyoto, Japan). The total RNA was treated with RQ RNase-free DNase (Promega, Madison, WI) to prevent genomic DNA contamination. The hypothalamus and purity of the total extracted RNA from the pituitary was examined with the ratio of 1.87 and 2.01 for A260/ A280 ratio. From 1 μg of the extracted total RNA, the reverse transcription reaction was performed to synthesize cDNA using the Transcriptor First strand cDNA Synthesis kit (Roche Diagnostics, Mannheim, Germany).
10
Isolation and Analysis of Uterine Cell RNA
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Total RNA was extracted from pooled UECs and USCs isolated from several mice using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA was treated with RQ RNase-free DNase (Promega, Madison, WI, USA) to remove any genomic DNA for 20 min at 25 °C, followed by 10 min at 75 °C to inactivate the DNase. RNA concentration and quality were assessed using a NanoDrop (ND-1000; Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from RNA using MMLV reverse transcriptase (BeamsBio, Seoul, Korea) and random hexamer primers (Invitrogen). Primers used for RT-PCR analyses are listed in Table 1 . Keratin 12 (Krt12) and desmin (Des) were used as markers of the uterine epithelium and stroma, respectively [21 (link)].
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