Illustra ready to go rt pcr beads

Manufactured by GE Healthcare

Sourced in United Kingdom

Illustra Ready-To-Go RT-PCR Beads are a pre-formulated reagent system for reverse transcription and real-time PCR. The beads contain all the necessary components for performing RT-PCR reactions, including reverse transcriptase, DNA polymerase, and dNTPs.

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Illustra™ Ready-to-GO RT-PCR beads kit (8 citations) PCR beads (3 citations) Illustra Ready-To-Go PCR Beads (2 citations)

13 protocols using illustra ready to go rt pcr beads

RNA-Seq Library Preparation and Sequencing

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Total RNA samples were first treated with DNAse-I to remove any possible DNA contamination, and then the mRNA was enriched using Dynabeads Oligo(dt)₂₅ (Thermo Fisher Scientific, USA). The enriched mRNA was fragmented in smaller fragments of 200 bps approximately, which were attached to adapters with known sequences that were unique for each sample. Samples were connected to magnetic beads containing complementary sequences for the adapters and then inserted in microwells where an emulsion-PCR for cDNA synthesis was carried (illustra Ready-To-Go RT-PCR Beads, GE Lifesciences). Our six cDNA libraries were submitted to quantification and quality control using Agilent 2100 Bioanalyzer and were then loaded in Ion Proton V2 PI chip using the Ion PI™ 200 Sequencing Kit v3 and sequenced using Ion Proton™ (Thermo Fisher Scientific, EUA) platform in a single multiplex run.
Raw data reads obtained by primary sequencing using Ion Proton™ were submitted to quality control to calculate alignment and to assess how the reads behave when compared to the reference human genome (Hg19/GRCh37). The aligned reads were mapped and quantified using TMAP (Torrent Mapping Alignment Program), which supports different alignment algorithms [30 (link)–32 (link)]. Processed datasets were uploaded to GEO (Gene Expression Omnibus) under the access number GSE81265.

Maués J.H., Ribeiro H.F., Pinto G.R., Lopes L.D., Lamarão L.M., Pessoa C.M., Moreira-Nunes C.D., de Carvalho R.M., Assumpção P.P., Rey J.A, & Burbano R.M. (2018). Gastric Cancer Cell Lines Have Different MYC-Regulated Expression Patterns but Share a Common Core of Altered Genes. Canadian Journal of Gastroenterology & Hepatology, 2018, 5804376.

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Quantitative RT-PCR Analysis of Gene Expression

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For gene expression analysis, total RNA from BMDMs was extracted using the TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. RNA was reverse transcribed (1 mg of total RNA) using Illustra Ready-To-Go RT-PCR Beads (GE Healthcare, Chicago, IL) as previously shown (Guimaraes et al., 2019 (link)). The PCR reaction was performed with QuantStudio3 real-time PCR instrument (Applied Biosystems) as previously described (Guimaraes et al., 2019 (link)). The primers were used to amplify a specific fragment corresponding to specific gene target: β-actin, forward, 5’-GGC TGT ATT CCC CTC CAT CG-3’, reverse, 5’-CCA GTT GGT AAC AAT GCC ATG T-3’; Galectin-3, forward, 5’-CAG GAA AAT GGC AGA CAG CTT-3′, reverse, 5’- CCC ATG CAC CCG GAT ATC-3′; IFN-β, forward, 5’-GCC TTT GCC ATC CAA GAG ATG C-3’, reverse, 5’-ACA CTG TCT GCT GGT GGA GTT C-3’; GBP2, forward, 5’-CTG CAC TAT GTG ACG GAG CTA-3’, reverse, 5’-CGG AAT CGT CTA CCC CAC TC-3’; GBP3, forward, 5’-CTG ACA GTA AAT CTG GAA GCC AT-3’, reverse, 5’-CCG TCC TGC AAG ACG ATT CA-3’; GBP4, forward, 5’-GGA GAA GCT AAC GAA GGA ACA A-3’, reverse, 5’-TTC CAC AAG GGA ATC ACC ATT TT-3’; GBP5, forward, 5’-CTG AAC TCA GAT TTT GTG CAG GA-3’, reverse, 5’-CAT CGA CAT AAG TCA GCA CCA G-3’. The results are presented as relative expression units after normalization to the β-actin gene. The experiments were conducted in triplicate.

Tana F.L., Guimarães E.S., Cerqueira D.M., Campos P.C., Gomes M.T., Marinho F.V, & Oliveira S.C. (2021). Galectin-3 regulates proinflammatory cytokine function and favors Brucella abortus chronic replication in macrophages and mice. Cellular microbiology, 23(10), e13375.

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Quantifying Gene Expression in Bone Marrow Macrophages

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Bone marrow-derived macrophages were homogenized with TRIzol reagent (Invitrogen) to isolate total RNA. Reverse transcription of 1 µg from total RNA was performed using illustra™ Ready-To-Go RT-PCR Beads (GE Healthcare, Buckinghamshire, UK). Real-time RT-PCR was conducted in a final volume of 10 µL containing the following: SYBR^® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), cDNA as the PCR template, and 20 µM of primers. The PCR reaction was performed with ABI 7900 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), using the following cycling parameters: 60°C for 10 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min, and a dissociation stage of 95°C for 15 s, 60°C for 1 min, 95°C for 15 s, and 60°C for 15 s. Primers were used to amplify a specific 100- to 120-bp fragment corresponding to specific gene targets as described in Table S1 in Supplementary Material. All data are presented as relative expression units compared with 0 h post-infection after normalization to the β-actin gene (ΔΔCt = ΔCt treatment − ΔCt 0 h post-infection) (26 (link)). PCR measurements were conducted in triplicate. The differences in the relative expression were analyzed by analysis of variance (ANOVA) followed by Tukey’s test (p < 0.05 for statistically significant).

Corsetti P.P., de Almeida L.A., Gonçalves A.N., Gomes M.T., Guimarães E.S., Marques J.T, & Oliveira S.C. (2018). miR-181a-5p Regulates TNF-α and miR-21a-5p Influences Gualynate-Binding Protein 5 and IL-10 Expression in Macrophages Affecting Host Control of Brucella abortus Infection. Frontiers in Immunology, 9, 1331.

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Quantitative RT-PCR for IFN-β Expression

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Reverse transcription of 1 µg from total RNA was performed using Illustra™ Ready-To-Go RT-PCR Beads (GE Healthcare, Little Chalfont, UK), following instructions of the manufacturer. Quantitative real-time PCR was conducted in a final volume of 10 µL containing the following: SYBR^® Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), oligo-dT cDNA as the PCR template, and 5 µM of primers. The PCR reaction was performed with StepOne Plus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA), using the following cycling parameters: 60°C for 10 min, 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min, and a dissociation stage of 95°C for 15 s, 60°C for 1 min, 95°C for 15 s, and 60°C for 15 s. Primers were used to amplify a specific 100–120 bp fragment corresponding to specific gene targets as follows: IFN-β, forward (5′-AGCTCCAAGAAAGGACGAACAT-3′); IFN-β, reverse (5′-GCCCTGTAGGTGAGGTTGATCT-3′); β-actin, forward (5′-AGGTGTGCACCTTTTATTGGTCTCAA-3′); β-actin, reverse (5′-TGTATGAAGGTTTGGTCTCCCT-3′). All data are presented as relative expression units after normalization to the β-actin gene. PCR measurements were conducted in triplicate.

Campos P.C., Gomes M.T., Guimarães E.S., Guimarães G, & Oliveira S.C. (2017). TLR7 and TLR3 Sense Brucella abortus RNA to Induce Proinflammatory Cytokine Production but They Are Dispensable for Host Control of Infection. Frontiers in Immunology, 8, 28.

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Molecular Techniques for Aspergillus DNA Extraction

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Standard molecular techniques are described by Sambrook et al. (1989) . Aspergillus genomic DNA was prepared as described by Jones and Sealy-Lewis (1989) (link). DNA was extracted from the strains [H3, H103, H44, H44(27), H44(23), H44(32), H7, H7 rev16] and PCR primers (Table S2) were designed to amplify overlapping sections of the entire coding region of the genes. The same primers were used for sequencing the PCR products. The coding region of both genes was sequenced on both strands. Where a change was identified compared with the sequence in the database, the wild-type was sequenced in that region to confirm the change. RT-PCR was performed using the GE Healthcare Illustra Ready-to-Go RT-PCR beads. The PCR products for eRF1 and the tubulin controls were standardly run on 1.3% TAE agarose gels or 5% TBE polyacrylamide gels. Crystal structure prediction of eRF1 and eRF3 was achieved through EsyPred3D Web server (http://www.fundp.ac.be/sciences/biologie/urbm/bioinfo/esypred/) and Swiss-model (http://swissmodel.expasy.org/).

Liu W., Mellado L., Espeso E.A, & Sealy-Lewis H.M. (2014). In Aspergillus nidulans the Suppressors suaA and suaC Code for Release Factors eRF1 and eRF3 and suaD Codes for a Glutamine tRNA. G3: Genes|Genomes|Genetics, 4(6), 1047-1057.

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Quantitative RT-PCR Analysis of iNOS Expression

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Total RNA from infected BMDMs was isolated using the TRIzol® reagent, accordingly to manufacturer instructions. Reverse transcription of 1 μg from total RNA was performed using illustra™ Ready-To-Go RT-PCR Beads (GE Healthcare, UK), following instructions of the manufacturer. Quantitative real-time PCR was conducted in a final volume of 10 μL containing the following: SYBR® Green PCR Master Mix (Thermo Fisher Scientific, USA), oligo-dT cDNA as the PCR template and 5 μM of primers. The PCR reaction was performed with QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, USA), using the following cycling parameters: 60°C for 10 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec and 60°C for 1 min, and a dissociation stage of 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec, 60°C for 15 sec. Primers were used to amplify a specific 100–120 base pairs fragment corresponding to specific gene targets as follows: iNOS Forward (5′-CAGCTGGGCTGTACAAACCTT-3′) and iNOS Reverse (5′-CATTGGAAGTGAAGCGTTTCG-3′); 18S Forward (5′-CGTTCCACCAACTAAGAACG-3′) and 18S Reverse (5′-CTCAACACGGGAAACCTCAC-3′). Data were analyzed using the threshold cycle (ΔΔC_T) method and they were presented as relative expression units after normalization to the 18S gene. PCR measurements were conducted in triplicate.

Campos P.C., Gomes M.T., Marinho F.A., Guimarães E.S., de Moura Lodi Cruz M.G, & Oliveira S.C. (2019). Brucella abortus nitric oxide metabolite regulates inflammasome activation and IL-1β secretion in murine macrophages. European journal of immunology, 49(7), 1023-1037.

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Murine Dermal Fibroblast RNA Isolation

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Twenty-four hours after treatment, total RNA was isolated from cultured murine dermal fibroblasts using a commercial kit (RNeasy Mini Kit, Qiagen) as described by the manufacturer. Two μg of RNA per sample were reverse transcribed using illustra Ready-To-Go RT-PCR Beads (GE Healthcare). Quantity and quality of total RNA and cDNA was assessed using Nanodrop 1000 (Thermo Scientific) and QIAxcel Advance system (Qiagen). The 7300 real time PCR system (Applied Biosystem, Life Technologies) was used to amplify cDNA using Power SYBR green mastermix (Applied Biosystems, Life Technologies). Sequences for primers used in all experiments and genotyping are provided in S1 Table.

Meyer P., Maity P., Burkovski A., Schwab J., Müssel C., Singh K., Ferreira F.F., Krug L., Maier H.J., Wlaschek M., Wirth T., Kestler H.A, & Scharffetter-Kochanek K. (2017). A model of the onset of the senescence associated secretory phenotype after DNA damage induced senescence. PLoS Computational Biology, 13(12), e1005741.

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Quantitative Gene Expression Analysis

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Expression levels of candidate genes were confirmed by qPCR. Total RNA (1 μg) was reverse transcribed using Illustra Ready-To-Go RT-PCR Beads (GE Healthcare Life Sciences, Buckinghamshire, UK) according to the manufacturer’s instructions. The qPCR reaction mixtures (20 μl) included 1 μl of each primer (10 μM), 10 μl of SYBR green PCR Master Mix (Roche Applied Science, Mannheim, Germany), and 5 μl of cDNA (10-fold diluted). Real-time qPCR was conducted by LightCycler® Nano System (Roche Applied Science, Mannheim, Germany). The thermal cycle was as follows: 95°C for 5 minutes, 45 cycles of 95°C for 10 seconds, annealing temperature (Ta) (Table 2) for 10 seconds, and 72°C for 15 seconds. The final melting curve analysis was performed at 65°C for 20 seconds, followed by 95°C for 20 seconds. Triplicates were done for each individual sample. The mRNA expression level of each target gene was normalized against elongation factor 1α (EF1α) expression level (a common reference gene). The normalized mRNA abundances were reported as the mean ± SE. The normalized mRNA abundances were reported as the mean ± SE. Specific primers were designed via Primer 3 [15 (link)] (Table 2). All of primers used for qPCR analysis were designed to span exon-exon junction.

Klangnurak W., Fukuyo T., Rezanujjaman M.D., Seki M., Sugano S., Suzuki Y, & Tokumoto T. (2018). Candidate gene identification of ovulation-inducing genes by RNA sequencing with an in vivo assay in zebrafish. PLoS ONE, 13(5), e0196544.

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Quantification of Guanylate-Binding Proteins

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RNA was extracted from BMDMs with TRIzol reagent (Invitrogen, Carlsbad, CA) to isolate total RNA in accordance with the manufacturer’s instructions. Reverse transcription of 2 μg of total RNA was performed using Illustra Ready-To-Go RT-PCR Beads (GE Healthcare, Chicago, IL) according to the manufacturer’s directions. Real-time RT-PCR was performed using 23 SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) on a QuantStudio3 real-time PCR instrument (Applied Biosystems, Foster City, CA). The appropriate primers were used to amplify a specific fragment corresponding to specific gene targets as follows: β-actin, forward, 5’- GGCTGTATTCCCCTCCATCG-3’, reverse, 5’-CCAGTTGGTAACAATGCCATGT-3’; GBP1, forward, 5’-GAGTACTCTCTGGAAATGGCCTCAGAAA-3’, reverse, TAGATGAAGGTGCTGCTGAGGAGGACTG-3; GBP2, forward, 5’-CTGCACTATGTG ACGGAGCTA-3’, reverse, 5’-CGG AATCGTCTACCCCACTC-3’; GBP3, forward, 5’-CTGACAGTAAATCTGGAAGCCAT-3’, reverse, 5’-CCGTCCTGCAAGACGATT CA-3’; GBP5, forward, 5’-CTGAACTCAGATTTTGTG CAGGA-3’, reverse, 5’-CATCGACATAAGTCAGCACCAG-3’; GBP7, forward, 5’-TCCTGTGTGCCTAGTGGAAAA-3’, reverse, 5’-CAAGCGGTTCATCAAGTAGGAT-3’. All data are presented as relative expression units after normalization to the β-actin gene, and measurements were conducted in triplicate.

Cerqueira D.M., Gomes M.T., Silva A.L., Rungue M., Assis N.R., Guimarães E.S., Morais S.B., Broz P., Zamboni D.S, & Oliveira S.C. (2018). Guanylate-binding protein 5 licenses caspase-11 for Gasdermin-D mediated host resistance to Brucella abortus infection. PLoS Pathogens, 14(12), e1007519.

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Quantification of Immune Response Genes

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RNA was extracted with Trizol reagent (Invitrogen) to isolate total RNA in accordance with the manufacturer’s instructions. Reverse transcription of 2 µg of total RNA was performed using Illustra Ready-To-Go RT-PCR Beads (GE Healthcare) according to the manufacturer’s instructions. Real-time RT-PCR was performed using 2x SYBR Green PCR Master Mix (Applied Biosystems) on an ABI 7900 real-time PCR instrument (Applied Biosystems). Appropriate primers were used to amplify the following specific fragments corresponding to specific gene targets: β-actin F: 5’-GGC TGT ATT CCC CTC CAT CG-3’; β-actin R: 5’-CCA GTT GGT AAC AAT GCC ATG T-3’; IFN- β F: 5’-GCC TTT GCC ATC CAA GAG ATG C-3’; IFN- β R: 5’-ACA CTG TCT GCT GGT GGA GTT C-3’; GBP2 F: 5’-CTG CAC TAT GT G ACG GAG CTA-3’; GBP2 R: 5’-CGG AAT CGT CTA CCC CAC TC-3’; GBP3 F: 5’-CTG ACA GTA AAT CTG GAA GCC AT-3’; GBP3 R: 5’-CCG TCC TGC AAG A CG ATT CA-3’; GBP5 F: 5’-CTG AAC TCA GAT TTT GTG CAG GA-3’; and G BP5 R: 5’-CAT CGA CAT AAG TCA GCA CCA G-3’. All data are presented as relative expression units after normalization to the β-actin gene, and measurements were conducted in triplicate.

Costa Franco M.M., Marim F.M., Alves-Silva J., Cerqueira D., Oliveira M.R., Tavares I.P, & Oliveira S.C. (2018). AIM2 senses Brucella abortus DNA in dendritic cells to induce IL-1β secretion, pyroptosis and resistance to bacterial infection in mice. Microbes and infection, 21(2), 85-93.

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