Superscript 3 first strand synthesis system kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, Australia

The SuperScript III First-Strand Synthesis System kit is a tool designed for the reverse transcription of RNA into complementary DNA (cDNA). It contains reagents and enzymes necessary for the reverse transcription process.

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Lab products found in correlation

137 protocols using superscript 3 first strand synthesis system kit

DRG and HEK-293 RNA Extraction and cDNA Synthesis

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DRG were dissected as previously described (refer to DRG extraction section) and were collected in an Eppendorf tube containing 200 μL/tube of RNA-Later solution. Tubes were left at −20 °C overnight and the day after tissue was crushed with a pestle and lysed by adding 200 μL/tube of freshly prepared lysis buffer (RNeasy Protect Mini Kit, Qiagen) and mercaptoethanol. RNA was extracted following standard procedure using the RNeasy Protect Mini protocol (Qiagen) including DNase I digestion step. Total RNA extracted was converted to first-strand cDNA using the SuperScript III First-Strand Synthesis System kit (Thermo Fisher Scientific). For HEK-293 cell experiment, cells were lysed by adding 600 μL/well of freshly prepared lysis buffer (RNeasy Protect Mini Kit, Qiagen) and mercaptoethanol. Cell lysates were transferred to Eppendorf tubes and RNA was extracted following standard procedure using the RNeasy Protect Mini protocol (Qiagen) including DNase I digestion step. Total RNA extracted was converted to first-strand cDNA using the SuperScript® III First-Strand Synthesis System kit (Thermo Fisher Scientific).

De Virgiliis F., Hutson T.H., Palmisano I., Amachree S., Miao J., Zhou L., Todorova R., Thompson R., Danzi M.C., Lemmon V.P., Bixby J.L., Wittig I., Shah A.M, & Di Giovanni S. (2020). Enriched conditioning expands the regenerative ability of sensory neurons after spinal cord injury via neuronal intrinsic redox signaling. Nature Communications, 11, 6425.

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Quantifying Gene Expression in Zika Virus Infection

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Total RNA from MOCK- or ZIKV-infected samples were extracted using the RNeasy Kit (Qiagen) according to the manufacturer’s instructions. Purified RNA was subjected to a reverse transcription using the SuperScript III first-strand synthesis system kit (Invitrogen). Gene expression was quantified by qRT-PCR using Light Cycler 480 SYBR green I master mix (Roche) and specific primers (Table 1). qRT-PCR were performed in 96-well plates and run on a Light Cycler 480 instrument (Roche). The housekeeping hypoxanthine-guanine phosphoribosyl transferase (HPRT) was used as control. The results are presented as fold change from matched mock-infected controls calculated using the 2^−ΔΔCT method.

Espino A., Gouilly J., Chen Q., Colin P., Guerby P., Izopet J., Amara A., Tabiasco J., Al-Daccak R., El Costa H, & Jabrane-Ferrat N. (2022). The mechanisms underlying the immune control of Zika virus infection at the maternal-fetal interface. Frontiers in Immunology, 13, 1000861.

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Polyploid CenH3 Expression Quantification

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Reverse transcription was performed using the Invitrogen SuperScript III First-Strand Synthesis System Kit using oligo dT primers, and CenH3 was amplified from the cDNA pool using primers that were designed from the 5’ and 3’ outermost exons (Additional file 6). PCR products were processed using the Qiaquick PCR Cleanup columns and sequenced with the amplification primers (Additional file 6).
Chromatogram-based expression estimates were calculated as described previously [78 (link)]. At least three replicates were used for each tissue to permit standard error calculations and paired, two-tailed T tests were used to test for significance. Expression levels for the polyploid accessions were secondarily estimated with RNA-seq data and by cloning cDNA amplicons (as described above). The clones were randomly selected from each sample, sequenced, and then grouped by their subgenomic origin “A_T” and “D_T”. Since the samples should follow a binomial distribution, the null hypothesis for the rate of cloning each homeologous copy of CenH3 should be 0.5. To control for the FWER (Family-wise Error Rate) at α = 0.05, the Bonferroni correction was determine the significance.

Masonbrink R.E., Gallagher J.P., Jareczek J.J., Renny-Byfield S., Grover C.E., Gong L, & Wendel J.F. (2014). CenH3 evolution in diploids and polyploids of three angiosperm genera. BMC Plant Biology, 14, 383.

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Quantitative Analysis of COL4A5 Transcripts

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Total RNA was extracted from cells using an RNA Isolation Kit (Zymo Research, Irvine, CA). RNA integrity was assessed after agarose gel electrophoresis, and the concentrations were measured spectrophotometrically (Nanodrop Technologies, Wilmington, DE). The samples were subjected to DNase treatment using a DNA-free kit (Ambion, Inc., Austin, TX), and 1 μg from each sample reverse-transcribed using oligo dT and a SuperScript III First Strand Synthesis System Kit (Invitrogen).
Samples were then assayed for COL4A5 transcripts, using the fluorescent intercalating agent SYBR Green 1 (Qiagen, Hilden, Germany), specific primers (Life Technologies, Mulgrave, Victoria, Australia) (Supplementary Tables S1 and S2), and the ABI 7500 real-time PCR System (Applied Biosystems, Waltham, MA). Individual reactions comprised 5 μl of 2× QuantiTect SYBR Green RT-PCR Master Mix (Qiagen), 0.7 μl each of 20 ng/μl sense and antisense primer, and 2 μl of 100 ng/μl cDNA template, in a total volume of 10 μl. The threshold cycle value was calculated at the end of each run using glyceraldehyde-3-phosphate dehydrogenase as the internal control and software provided by the manufacturer. Each sample was examined in duplicate and the assays performed in triplicate. The results were compared with expression in the 3 normal male fibroblast cell lines in different experiments.

Wang D., Mohammad M., Wang Y., Tan R., Murray L.S., Ricardo S., Dagher H., van Agtmael T, & Savige J. (2017). The Chemical Chaperone, PBA, Reduces ER Stress and Autophagy and Increases Collagen IV α5 Expression in Cultured Fibroblasts From Men With X-Linked Alport Syndrome and Missense Mutations. Kidney International Reports, 2(4), 739-748.

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Quantitative Real-Time PCR for Gene Expression

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Total RNA was extracted with Trizol (Invitrogen). 2 μg RNA were retro-transcribed using the SuperScript III First-Strand Synthesis System kit (Invitrogen). Gene expression was measured by quantitative real-time PCR using 7900 HT Fast Real-Time PCR System (Applied Biosystems). The level of each transcript was measured with the threshold cycle (Ct) method. Values were normalized to the mean of the cells expressing AR24Q or AR100Q, which were assigned as 100%.

Scaramuzzino C., Casci I., Parodi S., Lievens P.M., Polanco M.J., Milioto C., Chivet M., Monaghan J., Mishra A., Badders N., Aggarwal T., Grunseich C., Sambataro F., Basso M., Fackelmayer F.O., Taylor J.P., Pandey U.B, & Pennuto M. (2015). Protein Arginine Methyltransferase 6 Enhances Polyglutamine-Expanded Androgen Receptor Function and Toxicity in Spinal and Bulbar Muscular Atrophy. Neuron, 85(1), 88-100.

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Profiling Cytokine Expression in THP-1 Cells

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Total RNA from THP-1 monocytes and macrophages (1.2 × 10⁶ cells) was isolated by using the RNeasy mini kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. RNA concentration and purity were determined using a NanoDrop-8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Extracted RNA was used for cDNA synthesis using the SuperScript III First-Strand Synthesis System kit (Invitrogen, Thermo Fischer Scientific, Waltham, MA, USA) on a thermal cycler (T Professional Basic Gradient 96; Biometra, Goettingen, Germany). Real-time PCR was performed with a LightCycler 480 SYBR Green I Master kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions using a LightCycler 480 II Real-Time PCR System (Roche Diagnostics, Mannheim, Germany). The specific primers of human TNF-α, IL-1α, IL-6, IL-8, IL-10, CXCL10 and GAPDH genes are shown in Table 1. PCR condition was performed according to the manufacturer’s instructions. The Ct values were measured in triplicate. Gene expression was normalized to that of GAPDH mRNA in the same samples, using the 2^–∆∆Ct method where ΔCt represents the Ct (target gene)—Ct (GAPDH), and ΔΔCt represents the ΔCt (stimulated cells)—ΔCt (non-stimulated cells).

Lertjuthaporn S., Somkird J., Lekmanee K., Atipimonpat A., Sukapirom K., Sawasdipokin H., Tiewcharoen S., Pattanapanyasat K, & Khowawisetsut L. (2022). Extracellular Vesicles from Naegleria fowleri Induce IL-8 Response in THP-1 Macrophage. Pathogens, 11(6), 632.

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PBMC Transcription Profiling Assay

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1 × 10⁶ PBMCs were stimulated for 6, 12, 16, 24, or 48 h with or without PMA/Iono as described above. RNA was extracted with RNeasy® Mini Kit (QIAGEN). Seven microliters of extracted RNA was reverse transcribed with SuperScript™ III First-Strand Synthesis System kit (Invitrogen). qPCR was run with ssoAdvanced™ master-mix, RPL13a, SDHA, TBP and IFN-γ primers at 500 nM and samples at 10² cells/reaction.

Browne D.J., Brady J.L., Waardenberg A.J., Loiseau C, & Doolan D.L. (2020). An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells. Frontiers in Immunology, 11, 402.

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Embryonic Nasal Tissue Cryosectioning and RNA Extraction

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Ethical approval for the use of postmortem human foetal nasal tissue was given by the National Research Ethics Service Committee East of England - Cambridge Central, UK. The tissue was collected at Addenbrooke’s Hospital (Cambridge, UK) from patients who had requested pregnancy terminations, and dissected at the John van Geest Centre for Brain Repair, University of Cambridge, Cambridge, UK. Tissue comprising the cartilage of the nasal septum, the medial zygomatic region, the maxillae of the developing upper jaw, and the base of the orbital cavities was dissected in HIBERNATE media (Gibco) from the facial region of 7-8-week embryos.
For cryosectioning, the tissue was immersion-fixed overnight in 4% paraformaldehyde in PBS at 4°C, then cryoprotected in 30% diethylpyrocarbonate-treated sucrose, embedded in O.C.T. compound (Tissue Tek), flash frozen in isopentane on dry ice and stored at -80°C. 10 μm sections were taken on a rotary cryostat (Bright Instrument Company).
For cDNA synthesis, the tissue was transferred to the lysis buffer from the Arcturus PicoPure RNA extraction kit (ThermoFisher Scientific), and then to Trizol (Invitrogen) for homogenisation and total RNA extraction according to the manufacturer's instructions. Single-strand cDNA was generated using Invitrogen’s Superscript III First-Strand Synthesis System kit.

Miller S.R., Perera S.N., Benito C., Stott S.R, & Baker C.V. (2016). Evidence for a Notch1‐mediated transition during olfactory ensheathing cell development. Journal of Anatomy, 229(3), 369-383.

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Watermelon Leaf RNA Extraction

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Infected and control watermelon leaves were ground to a powder in liquid nitrogen and 100 mg of each sample was subjected to total RNA extraction using an RNeasy Mini kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. First-strand cDNA was synthesized from total RNA with a SuperScript III First-Strand Synthesis System kit (Invitrogen, Gaithersburg, MD).

Hassan M.Z., Rahim M.A., Jung H.J., Park J.I., Kim H.T, & Nou I.S. (2019). Genome-Wide Characterization of NBS-Encoding Genes in Watermelon and Their Potential Association with Gummy Stem Blight Resistance. International Journal of Molecular Sciences, 20(4), 902.

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RNA Isolation and cDNA Synthesis

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RNA isolation and complementary DNA preparation were using the RNeasy Mini Kit (163037260, QIAGEN) and the SuperScript III First-Strand Synthesis System Kit (Invitrogen) following the manufacturer’s protocol, respectively. The target genes expression was verified by quantitative PCR (qPCR) with KAPA SYBR FAST qPCR Master Mix (KK4600), and glyceraldehyde-3-phosphate dehydrogenase was used as a control.

Wang J., Huang T.Y., Hou Y., Bartom E., Lu X., Shilatifard A., Yue F, & Saratsis A. (2021). Epigenomic landscape and 3D genome structure in pediatric high-grade glioma. Science Advances, 7(23), eabg4126.

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