Superscript 3 first strand cdna synthesis system

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The SuperScript III First-Strand cDNA Synthesis System is a laboratory product used for the reverse transcription of RNA into complementary DNA (cDNA). It provides a streamlined workflow for the synthesis of first-strand cDNA from total RNA or poly(A)+ RNA templates.

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55 protocols using superscript 3 first strand cdna synthesis system

Quantitative RT-PCR Analysis of Plant Transcripts

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Total RNA from maize or Arabidopsis was extracted using RNeasy Plant Mini Kit (Qiagen, Germantown, MD, United States) and treated with rDNaseI (Thermo Fisher, Waltham, MA, United States) per manufacturers’ instructions. First strand cDNA was synthesized from 1 μg of total RNA and oligo (dT)₂₀ primers using Superscript III First Strand cDNA synthesis system (Invitrogen, Carlsbad, CA, United States). For qRT-PCR analysis, SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, United States) was used on an Applied Biosystems Mx3000P thermocycler with 10 ng (for maize) or 5 ng (for Arabidopsis) of cDNA as template in a 10 μL reaction. Primer sequences are described in Supplementary Table 1. Relative expression levels compared to internal reference ZmACTIN (Louis et al., 2015 (link)) or AtEF1α-A (Aboobucker et al., 2017 (link)) were calculated using the 2^–ΔΔCt method (Pfaffl, 2001 (link)).

Aboobucker S.I., Showman L.J., Lübberstedt T, & Suza W.P. (2021). Maize Zmcyp710a8 Mutant as a Tool to Decipher the Function of Stigmasterol in Plant Metabolism. Frontiers in Plant Science, 12, 732216.

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Quantitative Analysis of Antiviral Gene Expression in SARS-CoV-2-Infected Cells

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Total RNA was extracted from THP-1 cells and various other cell types with the Quick-RNA MicroPrep kit (Zymo Research). RNA was reverse-transcribed with random hexamers and the Superscript III first-strand cDNA synthesis system (Invitrogen). Quantitative real-time PCR was then performed with the TaqMan universal PCR master mix (Applied Biosystems). For gene expression assays, TaqMan probes for OAS1, OAS2, OAS3, RNASEL, IL6, and CXCL9 were used (Thermo Fisher Scientific). We used β-glucuronidase (GUSB) for normalization (Applied Biosystems). The results were analyzed with the ΔCt or ΔΔCt method. For SARS-CoV-2 genomic RNA quantification, RNA was extracted from 3 × 10⁵ THP-1 cells infected with SARS-CoV-2 for 24 hours. Cells were washed three times with PBS and lysed for RNA extraction. Equal amounts of total RNA were reverse-transcribed with random hexamers and the Superscript III first-strand cDNA synthesis kit (Invitrogen). Equal amounts of cDNA were used for the qPCR reaction. Primers and probes for the N gene (N2 region), the RNA-dependent RNA polymerase (RdRP) gene, and their respective standards were purchased from IDT technologies. All qPCR reactions were analyzed with the QuantStudio 3 system.

Boyce F.M., Beggs A.H., Feener C, & Kunkel L.M. (1991). Dystrophin is transcribed in brain from a distant upstream promoter.. Proceedings of the National Academy of Sciences of the United States of America, 88(4), 1276-1280.

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Nrf2, HO-1, and NQO1 Gene Expression

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JB6 P+ cells were seeded in 6-cm diameter dishes at a density of 1 × 10⁴ cells/dish. After incubation for 24 h, the cells were treated with TAX at different concentrations for five days. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). The SuperScript III First-Strand cDNA Synthesis System (Invitrogen, Waltham, MA, USA) was used to synthesize first-strand cDNA. The mRNA expression of Nrf2, HO-1 and NQO1 was determined using quantitative reverse transcription-PCR (qRT-PCR). The sequences of the primers for Nrf2, HO-1 and NQO1 were used as shown in Table 1.

Kuang H., Tang Z., Zhang C., Wang Z., Li W., Yang C., Wang Q., Yang B, & Kong A.N. (2017). Taxifolin Activates the Nrf2 Anti-Oxidative Stress Pathway in Mouse Skin Epidermal JB6 P+ Cells through Epigenetic Modifications. International Journal of Molecular Sciences, 18(7), 1546.

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Quantitative Analysis of Gene Expression in Colon Epithelial Cells

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RNA extracted from colon epithelial cells or cell lines was reverse-transcribed into complementary DNA (cDNA) using the SuperScriptIII First Strand cDNA synthesis system (Invitrogen). cDNA was synthesized from 0.5 µg RNA using random hexamer primers and SuperScriptIII (Invitrogen). Real-time reverse-transcriptase-polymerase chain reaction (RT-PCR) was performed on a Bio-Rad iCycler to quantify messenger RNA (mRNA) levels. The primers are as follows: GM-CSF—sense: 5′-TCT CAG CAC CCA CCC GCT CA-3′, anti-sense: 5′-GCC CCG TAG ACC CTG CTC GAA-3′; Arg-1—sense: 5′-CTC CAA GCC AAA TAC AAG A-3′, anti-sense: 5′-AGG AGC TGT CAT TAG GGA CAT C-3′; iNOS—sense: 5′-GTT CTC AGC CCA ACA ATA CAA GA-3′, anti-sense: 5′-GTG GAC GGG TCG ATG TCA C-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)—sense: 5′-TCT TGG GCT ACA CTG AGG AC-3′, anti-sense: 5′-CAT ACC AGG AAA TGA GCT TGA-3′. All reactions were performed in triplicate. The data were analyzed using Q-Gene software and expressed as fold change mean normalized expression (MNE) from control value. MNE is directly proportional to the amount of RNA of the target gene relative to the amount of RNA of the reference gene, GAPDH.

Ma N., Liu Q., Hou L., Wang Y, & Liu Z. (2017). MDSCs are involved in the protumorigenic potentials of GM-CSF in colitis-associated cancer. International Journal of Immunopathology and Pharmacology, 30(2), 152-162.

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Nucleic Acid Extraction and Quantification

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Nucleic acids were extracted from filter samples using the NucleoMag plant kit (Macherey-Nagel) for genomic DNA (gDNA) and the NucleoMag RNA kit (Macherey-Nagel) for RNA. Initial sample lysis buffer resuspension and vortexing was completed manually, the remainder using an epMotion liquid handling system (Eppendorf). gDNA was quantified using the Quant-iT PicoGreen double-stranded DNA assay kit (Invitrogen), and RNA was quantified using the Quant-iT RiboGreen RNA assay kit. Nucleic acid integrity was confirmed using an Agilent 2200 TapeStation (Agilent). RNA was reverse-transcribed into cDNA using the SuperScript III first-strand cDNA synthesis system (Invitrogen). Genomic DNA was extracted from Vibrio-enriched pellets using a DNeasy blood and tissue kit (Qiagen), with subsequent quantification and quality control as described above.

Diner R.E., Kaul D., Rabines A., Zheng H., Steele J.A., Griffith J.F, & Allen A.E. (2021). Pathogenic Vibrio Species Are Associated with Distinct Environmental Niches and Planktonic Taxa in Southern California (USA) Aquatic Microbiomes. mSystems, 6(4), e00571-21.

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Validating Gene Expression in Lonicera Flower Buds

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Quantitative reverse transcription-PCR (qRT-PCR) was utilized to validate the patterns of gene expression in the flower buds of L. japonica under light intensity treatments. Total RNA was isolated using the same method mentioned above, and then the first-strand cDNA was synthesized using the Superscript® III First Strand cDNA Synthesis System (Invitrogen, USA) following the manufacturer’s protocol. Actin was used as a reference for calculating the gene expression normalization with the

2^{- Δ Δ C_{t}}

method (Livak and Schmittgen 2001 (link)). The gene-specific primers were designed with the software Primer Premier 5.0 and are shown in Table S1. The qRT-PCR was performed using SYBR Premix Ex Taq™ II kit (Takara) as per manufacturer’s instructions, and conducted in qTOWER 2.2 Real-Time PCR System (Analytik Jena AG, Jena, Germany) under the following parameters: 94 °C for 2 min, followed by 40 cycles of 94 °C for 20 s, and 60 °C for 34 s. All reactions were performed in three biological replicates and three technical replicate, and the results were reported as mean ± standard error.

Fang H., Qi X., Li Y., Yu X., Xu D., Liang C., Li W, & Liu X. (2019). De novo transcriptomic analysis of light-induced flavonoid pathway, transcription factors in the flower buds of Lonicera japonica. Trees (Berlin, Germany : West), 34(1), 267-283.

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Isolation and Analysis of Naive Splenic CD4+ T Cells

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Naive splenic CD4⁺ T cells were isolated by a Naive CD4⁺ T Cell Isolation Kit (Miltenyi Biotec GmbH) and then were lysed in TRIzol™ Reagent (Invitrogen). Isolated total RNA using a phenol-chloroform extraction was reverse-transcribed with the SuperScript III First-Strand cDNA Synthesis System (Invitrogen). RT-PCRs were performed on the ABI PRISM 7300 Real-Time PCR System (Applied Biosystem) using FastStart SYBR Green Master Mix (Roche Applied Science). Primers used were as follows: Ccl1 (forward, 5′-TTCCCCTGAAGTTTATCCAGTGTT-3′; reverse, 5-′TGAACCCACGTTTTGTTAGTTGAG-3′), Ccl2 (forward, 5′-GTCCCTGTCATGCTTCTGGG-3′; reverse, 5′-GCGTTAACTGCATCTGGCTG-3′), Ccl3 (forward, 5′-CCAGCCAGGTGTCATTTTCC-3′; reverse, 5′-CTCAAGCCCCTGCTCTACAC-3′), Il-6 (forward, 5′-GAGGATACCACTCCCAACAGACC-3′; reverse, 5′-AAGTGCATCATCGTTGTTCATACA-3′), Tnf (forward, 5′-CCCTCACACTCAGATCATCTTCT-3′; reverse, 5′- GCTACGACGTGGGCTACAG-3′), Csf1 (forward, 5′-GGTCCTGCAGCAGTTGATCG-3′; reverse, 5′-CTCGGTGGCGTTAGCATTGG-3′), Gzmb (forward, 5′-GACCCAAAGACCAAACGTGC-3′; reverse, 5′-TCTGTAGTTAGCTGCTTTTCATTGT-3′), and Gzmk (forward, 5′-CCGCCCACTGCTACTCTTG-3′; reverse, 5′-TGCAGCAGTGCGAAGCTTTATC-3′).

Xu L., Wei C., Chen Y., Wu Y., Shou X., Chen W., Lu D., Sun H., Li W., Yu B., Wang X., Zhang X., Yu Y., Lei Z., Tang R., Zhu J., Li Y., Lu L., Zhou H., Zhou S., Su C, & Chen X. (2022). IL-33 induces thymic involution-associated naive T cell aging and impairs host control of severe infection. Nature Communications, 13, 6881.

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Measuring mRNA Levels in E. coli

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The procedure for measuring mRNA level was described before^{46 (link)}. Briefly, E. coli was grown in modified M9 minimal medium until reaching the exponential phase. Cells were harvested in RNase-free vials and centrifuged for 10,000 rpm for 10 min to obtain the cell pellet. Total RNA was extracted using an RNA isolation kit (Macherey–Nagel, Germany). First-strand cDNA was synthesized using a SuperScript III first-strand cDNA synthesis system (Invitrogen, USA). RT-PCR was performed by the StepOne real-time PCR system (Applied Biosystems, USA). The thermal cycling conditions were as follows: denaturation, 1 cycle of 95 °C for 30 s; and amplification, 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The relative mRNA level was calculated using the ΔC_t method. The housekeeping gene rpoD encoding sigma factor 70 was used as a reference.

Nguyen-Vo T.P., Ko S., Ryu H., Kim J.R., Kim D, & Park S. (2020). Systems evaluation reveals novel transporter YohJK renders 3-hydroxypropionate tolerance in Escherichia coli. Scientific Reports, 10, 19064.

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Quantifying Gene Expression by RT-PCR

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A two-step RT-PCR reaction was employed. cDNA was synthesized according to the protocol of the Superscript III First-Strand cDNA
Synthesis System (Invitrogen, Carlsbad, CA, USA) using 1 µg of total RNA. cDNA was diluted, and frozen aliquots were stored at
−20°C. The cDNA generated was amplified using SYBR^® Premix Ex Taq^TM II (Tli RNaseH Plus) (Takara Bio, Inc.,
Shiga, Japan). All assays were performed on a TP800 Thermal Cycler Dice^® Real Time System (Takara Bio, Inc.). Primers
were prepared for amplification of specific genes, TNF, CRLF2, GAPDH, CD86, STAT4 and IL-8 genes. The sequences of the primers
(forward and reverse primers) used in PCR reactions were 5′-AGATGATCTGACTGCCTGGG-3′ and 5′-CTGCTGCACTTTGGAGTGAT-3′ for TNF,
5′-CTGATGCCACGAAAATCTCA-3′ and 5′-TTCTCCATCAGGAATGGGAC-3′ for CRLF25’, 5′-GCACCGTCAAGGCTGAGAAC-3′ and 5′-TGGTGAAGACGCCAGTGGA -3′
for GAPDH, 5′-AGAGGAGCAGCACCAGAGAG-3′ and 5′-CAGAAGCAGCCAAAATGGAT-3′ for CD86, 5′-CACAGCTACATGCATTGGATT-3′and
5′-CGTGTTTCCAAAGAGAAAAACC-3′ for STAT4 and 5′-CTGGCCGTGGCTCTCTTG-3′ and 5′-CCTTGGCAAAACTGCACCTT-3′ for and IL8. A cycle threshold
(Ct) was assigned at the beginning of the logarithmic phase of PCR amplification. Data were analyzed by ABI software, and the gene
expression was quantified using the 2^−ΔΔCT method and normalized to the constitutively expressed housekeeping gene
GAPDH.

YANAGIHARA S., GOTO H., HIROTA T., FUKUDA S., OHNO H, & YAMAMOTO N. (2014). Lactobacillus acidophilus L-92 Cells Activate Expression of Immunomodulatory Genes in THP-1 Cells. Bioscience of Microbiota, Food and Health, 33(4), 157-164.

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Quantitative Analysis of Antiviral Gene Expression in SARS-CoV-2-Infected Cells

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Lee D., Pen J.L., Yatim A., Dong B., Aquino Y., Ogishi M., Pescarmona R., Talouarn E., Rinchai D., Zhang P., Perret M., Liu Z., Jordan I., Bozdemir S.E., Bayhan G.I., Beaufils C., Bizien L., Bisiaux A., Lei W., Hasan M., Chen J., Gaughan C., Asthana A., Libri V., Luna J.M., Jaffré F., Hoffmann H.H., Michailidis E., Moreews M., Seeleuthner Y., Bilguvar K., Mane S., Flores C., Zhang Y., Arias A.A., Bailey R., Schlüter A., Milisavljevic B., Bigio B., Voyer T.L., Materna M., Gervais A., Moncada-Velez M., Pala F., Lazarov T., Levy R., Neehus A.L., Rosain J., Peel J., Chan Y.H., Morin M.P., Pino-Ramirez R.M., Belkaya S., Lorenzo L., Anton J., Delafontaine S., Toubiana J., Bajolle F., Fumadó V., DeDiego M.L., Fidouh N., Rozenberg F., Pérez-Tur J., Chen S., Evans T., Geissmann F., Lebon P., Weiss S.R., Bonnet D., Duval X., Pan-Hammarström Q., Planas A.M., Meyts I., Haerynck F., Pujol A., Sancho-Shimizu V., Dalgard C.L., Bustamante J., Puel A., Boisson-Dupuis S., Boisson B., Maniatis T., Zhang Q., Bastard P., Notarangelo L., Béziat V., de Diego R.P., Rodriguez-Gallego C., Su H.C., Lifton R.P., Jouanguy E., Cobat A., Alsina L., Keles S., Haddad E., Abel L., Belot A., Quintana-Murci L., Rice C.M., Silverman R.H., Zhang S.Y, & Casanova J.L. (2023). Inborn errors of OAS–RNase L in SARS-CoV-2–related multisystem inflammatory syndrome in children. Science (New York, N.Y.), 379(6632), eabo3627.

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