RNA was isolated from the cultured cells using the innuPREP RNA Mini Kit (Analytik Jena). RNA concentration was quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific), and cDNA was synthesized using the SensiFAST cDNA synthesis kit (Bioline GmbH). SensiFAST SYBR No-ROX (Roche) reagent together with gene-specific primers (Table S2 of supporting information 1 ) were used for gene expression quantification. PCR products of each gene were initially cloned in the pGEM-T vector (Promega) and sequenced to verify the specificity of the primer pairs before analysis. Five different dilutions of cloned plasmids were used as standards and amplified together with cDNA samples in each run. The PCR amplification was performed in duplicates for each sample by taking 2.5 and 5 μl of cDNA in a total volume of 12 μl of reaction mix using a Light Cycler 96 instrument (Roche). PCR amplicon was verified for each run using melting curve analysis and agarose gel electrophoresis of PCR products.
Innuprep rna mini kit
Manufactured by Analytik Jena
Sourced in Germany
The InnuPREP RNA Mini Kit is a laboratory equipment designed for the isolation and purification of total RNA from a variety of sample materials, including cells, tissues, and microorganisms. The kit utilizes a spin column-based technology to efficiently extract and concentrate RNA, making it suitable for downstream applications such as reverse transcription and real-time PCR analysis.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (17 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (12 mentions)
M mlv reverse transcriptase, by Promega (10 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (10 mentions)
Maxima h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (9 mentions)
Gotaq qpcr master mix, by Promega (8 mentions)
Oligo dt 15 primer, by Promega (7 mentions)
Primescript rt master mix, by Takara Bio (7 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (7 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (7 mentions)
Quantstudio 3, by Thermo Fisher Scientific (6 mentions)
Revertaid h minus reverse transcriptase, by Thermo Fisher Scientific (6 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
Sensifast cdna synthesis kit, by Meridian Bioscience (5 mentions)
Iscript cdna synthesis kit, by Bio-Rad (5 mentions)
Pgem t vector, by Promega (4 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (4 mentions)
139 protocols using innuprep rna mini kit
1
Quantitative PCR Analysis of Gene Expression
2
Quantitative Real-Time PCR Analysis of Gene Expression
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Following treatments, cells were washed in PBS (ThermoScientific, Waltham, USA), total RNA was extracted using the innuPREP RNA Mini kit (Analytikjena, Berlin, Germany) by following the supplier recommendations. RNA concentration and quality preparations were determined using a NanoDrop 2000 (ThermoScientific). cDNA synthesis was performed in of total RNA (500 ng) using the RevertAid reverse transcription kit (ThermoScientific) and an oligo(dT)12−18 primer according to the supplier's instructions. Target genes were then amplified using GoTaq qPCR Master Mix (Promega, Madison, USA) in Applied Biosystems 7500 fast Real-Time PCR System (ThermoScientific).
All experiments were performed independently with at least three biological repeats and technical replicates. Data were analyzed by the comparative Ct method, and expressed as fold change relative to control group. The pairs of primers for human target genes were sourced from Primer bank and synthesized by Sigma-Aldrich (Hamburg, Germany). Sequences of human primers used in the study are summarized inTable 1 .
All experiments were performed independently with at least three biological repeats and technical replicates. Data were analyzed by the comparative Ct method, and expressed as fold change relative to control group. The pairs of primers for human target genes were sourced from Primer bank and synthesized by Sigma-Aldrich (Hamburg, Germany). Sequences of human primers used in the study are summarized in
3
Quantification of miR-375 and Target Genes
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ST cells transfected with the miR-375 mimics, miR-375 inhibitor and miRNA-ShNC were harvested at 24 h post-transfection. The reverse transcription primers and fluorescence-labeled primers for quantitative analysis of miR-375 and its target genes were designed using Primer 5.0. All of the primers were synthesized by Shanghai Sangon (Table 1 ). Total RNA was extracted using the innuPREP RNA Mini Kit (Analytikjena, Germany) according to the manufacturer’s protocols. Based on the quality and concentration of RNA, cDNA was reversed transcribed with the PrimeScript RT Reagent Kit (Takara, Dalian, China) according to the manufacturer’s instructions. Quantitative polymerase chain reaction (qPCR) was performed using a SYBR Premix Ex Taq (TaKaRa, Japan) on a stratagene Mx3005P (Agilent, USA) according to the instructions. The qPCR volume was 20 μL. The relative mRNA levels of miR-375, RLF, HIGD1A, colorectal cancer associated 2 (COLCA2), POU class 3 homeobox 1 (POU3F1), and WW domain binding protein 1 like (WBP1L) were normalized to U6 and β-actin. The data were analyzed using the SPSS 19.0 software, and the fold change of expression was calculated using the 2−ΔΔCT method according to the following formula:
Here, 2−ΔΔCT refers to the relative expression ratio.
Here, 2−ΔΔCT refers to the relative expression ratio.
4
Quantitative Real-Time PCR Analysis of Pseudomonas stutzeri
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Total RNA was isolated with an innuPREP RNA minikit (Analytik Jena) and was reverse-transcribed into cDNA, which was diluted to 100 ng μl−1. Quantitative real-time PCR (qRT-PCR) assays were performed using total RNA preparations obtained from three independent cultures (three biological replicates). The gene-specific primers listed in Table S3 in the supplemental material were designed based on the full genome sequence of P. stutzeri A1501, and the 16S rRNA gene was used as the endogenous reference gene to normalize the expression of target genes in each cDNA template. The relative mRNA concentration was calculated by the comparative threshold cycle (2−ΔΔCT) method. The target gene copy numbers were determined in triplicate using the 7500 real-time PCR system and ChamQ SYBR qPCR master mix. All procedures were carried out according to the manufacturers’ recommendations. Data were analyzed using ABI Prism 7500 sequence detection system software (Applied Biosystems).
5
Quantitative PCR Analysis of OA Chondrocytes
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Isolation of total RNA, cDNA synthesis, and SYBR-green-based RT-qPCR experiments were performed as described50 . In brief, total RNA was extracted from OA chondrocytes cultivated in 12-well plates using the innuPREP RNA Mini Kit (Analytic Jena), and each sample was examined for quantity and quality on NanoDrop 1000 and Agilent 2100 Bioanalyzer Nano LabChip (Agilent Technologies) prior to reverse transcription into cDNA. RNA integrity numbers were between 9.5 and 10. The protocols strictly followed the minimal guidelines for the design and documentation of qPCR experiments. A detailed qPCR checklist containing all relevant information is provided by the authors upon request. mRNA levels were calculated as quantities relative to the untreated control group, considering amplification efficiencies and normalization to succinate dehydrogenase complex, subunit A (SDHA), which had been identified as stable reference gene under the experimental conditions of this study.
6
Quantitative Gene Expression Analysis in Rat Brain
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Total RNA was isolated from rat brain using the innuPREP RNA Mini Kit (Analytik Jena AG, Germany). The mRNA concentration was determined on a DS-11 Spectrophotometer/Fluorometer (DeNovix, USA). The Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., USA) was used to synthesize cDNA samples for subsequent RT-PCR on a Standard real-time PCR Thermal Cycler (Analytik Jena AG, Germany). Specific primer sequences for Vdr, Vdbp, Cyp27b1, Cyp24a1, Nf-κb, Iκb-alpha, and the glyceraldehyde 3-phosphate dehydrogenase (Gapdh) reference gene were designed using Primer BLAST software and used at a working concentration of 10 μM:
Target genes were amplified for 60 cycles using Maxima SYBER Green/ROX qPCR Master Mix (Thermo Fisher Scientific Inc., USA). Relative mRNA expression calculations were performed according to the 2–ΔΔCt comparison method. The expression level of each gene was normalized for GAPDH in the same samples and then calculated as a fold change compared to the control.
Target genes were amplified for 60 cycles using Maxima SYBER Green/ROX qPCR Master Mix (Thermo Fisher Scientific Inc., USA). Relative mRNA expression calculations were performed according to the 2–ΔΔCt comparison method. The expression level of each gene was normalized for GAPDH in the same samples and then calculated as a fold change compared to the control.
7
Quantifying nifH mRNA Stability
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The stability of the nifH mRNA was determined in A1501 and the Δhfq or Δhfq (pLhfq) strains grown under nitrogen fixation conditions for 6 h followed by the addition of rifampicin (400 μg/mL, final concentration). Aliquots were added to two volumes of RNA protect (Sigma). After incubating at room temperature for 5 min, the samples were centrifuged for 5 min at 4°C and 12,000 rpm, and the pellets were quickly frozen in liquid nitrogen and stored at −80°C until they were ready for use. Total RNA was isolated with an innuPREP RNA Mini Kit (Analytik Jena) according to the manufacturer’s instructions. Full-length cDNAs were synthesized from total RNA using a First Strand cDNA synthesis kit (Takara Bio) and were used to estimate mRNA levels with qRT-PCR. The primers used to measure the mRNA half-life are listed in Table S4 (RT nifH F/RT nifH R). The relative mRNA concentration was calculated by the comparative threshold cycle (2−ΔΔCT) method with 16S rRNA as the endogenous reference. Data are presented as the percentage of nifH transcript levels relative to the amount of these transcripts at time point zero.
8
Transcriptome Sequencing of Spodoptera frugiperda
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For transcriptome sequencing, third to fourth instar larvae of S. frugiperda were fed maize leaves (2 weeks old plants, L4 stage) for 48 h, followed by dissection on cold phosphate buffer (pH 7.0, 10 mM). Guts and integument were collected separately, gut lumen briefly washed and stored in RNAlater (Sigma-Aldrich, Germany) overnight at 4°C before RNA extraction. Tissue samples from two individuals were pooled together for RNA extraction. Samples of Sf9 cell cultures (obtained during sub-culturing at full confluency) were centrifuged at 500 g for 5 min, the culture medium was discarded, and the fresh pellets were directly used for RNA extraction. Total RNA was extracted from tissues and Sf9 cell samples using the innuPREP RNA Mini Kit (Analytik Jena, Germany), followed by DNase treatment. RNA concentrations were measured with the NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific). First Strand cDNA was synthesized from 1 μg total RNA using SuperScript III Reverse Transcriptase and OligodT primers from Invitrogen.
9
Quantitative Analysis of hTERT Expression
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The expression of hTERT was evaluated using quantitative real-time reverse transcription PCR (qPCR). Total RNA was isolated from the cells using the innuPREP RNA Mini Kit (Analytik Jena, Jena, Germany) and reverse transcribed with the RevertAid RT Kit (Thermo Scientific, Thermo Fisher Scientific, Carlsbad, CA, USA), both according to the respective manufacturer’s instructions (innuPREP RNA Mini Kit, HB_KS-2080_e_170719, July 2017; RevertAid RT Kit User Guide). The qPCR was performed on a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific, Carlsbad CA, USA). Reaction primers with the following sequence were used to detect an amplicon of 149 base pairs: 5′-ACCGGAAGAGTGTCTGGAGCAA-3′, 5′-GGGGATGAAGCGGAGTCTGGA-3′. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5′-AGCTCACTGGCATGGCCTTC-3′, 5′-ACGCCTGCTTCACCACCTTC-3′) and β-actin (5′-ACTCTTCCAGCCTTCCTTC-3′, 5′-GATGTCCACGTCACACTTC-3′) were both used as reference genes for normalization, and the hTERT expression levels were quantified with the ΔCt method.
10
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was isolated from MEFs using an innuPREP RNA Mini Kit (Analytik-Jena). Reverse transcription and cDNA synthesis was carried out with 2 µg RNA using 200 U RevertAid H Minus Reverse Transcriptase, 100 pmol Oligo(dT)18, and 20 U RNase inhibitor (Thermo Fisher Scientific). Quantitative real-time PCR reactions employing SYBR Green were run in a 7900HT Fast Real-Time PCR System (Applied Biosystems). The following primers were used: 5′-GAGTCCGCAGCAGGTG -3′ and 5′-GTGTCAGAGTCCATGGGA -3′ for Xbp1s, 5′-GTGTCAGAGTCCATGGGA -3′ and 5′-GTGTCAGAGTCCATGGGA -3′ for Xbp1u, and 5′-CCCACTCTTCCACCTTCGATG -3′ and 5′-GTCCACCACCCTGTTGCTGTAG -3′ for Gapdh. Primers for the amplification of IE1 (M123), E1 (M112), gB (M55), and M37 transcripts have been described previously (Chapa et al., 2013 (link); Chapa et al., 2014 (link)). Reactions were performed under the following conditions: 45 cycles of 3 s at 95°C and 30 s at 60°C. Three replicates were analyzed for each condition, and the relative amounts of mRNAs were calculated from the comparative threshold cycle (Ct) values by using Gapdh as reference.
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