Superscript 3 rt kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Superscript III RT kit is a reverse transcription kit designed for the synthesis of first-strand cDNA from RNA templates. It includes the Superscript III reverse transcriptase enzyme, which is suitable for RT-PCR applications.

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179 protocols using superscript 3 rt kit

Quantification of mRNA and pre-mRNA levels

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Worm grinds of P0 (RNAi+) and F1, F2, F3 (RNAi−) generations were used for total RNA extraction with Trizol reagent (Life Technologies), followed by DNase I (NEB) treatment.
mRNA reverse transcription (RT). 1 μg of total RNA was used for the first-strand cDNA synthesis with SuperScript III RT kit (Life Technologies) and oligo(dT)₂₀ primer (to enrich for mRNA).
Pre-mRNA RT. 2 μg of total RNA was used for the first-strand cDNA synthesis with SuperScript III RT kit (Life Technologies) and random hexamer primer mix (to capture pre-mRNA).
qRT-PCR. qRT-PCR was performed using KAPA SYBR FAST Universal 2× PCR Master Mix (KAPA Biosystems) on a Mastercycler EP Realplex real-time PCR system (Eppendorf) according to the manufacturer’s instructions. qPCR primers are listed in Additional file 9: Table S2. Each sample was processed in triplicate. Reported values for the fold change of mRNA and pre-mRNA at oma-1 gene were calculated using ∆∆CT analysis.

Kalinava N., Ni J.Z., Peterman K., Chen E, & Gu S.G. (2017). Decoupling the downstream effects of germline nuclear RNAi reveals that H3K9me3 is dispensable for heritable RNAi and the maintenance of endogenous siRNA-mediated transcriptional silencing in Caenorhabditis elegans. Epigenetics & Chromatin, 10, 6.

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Quantitative Gene Expression Analysis

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For quantifying gene expression, RNA samples were extracted using TRIzol (Thermo Fisher Scientific). First-strand synthesis was conducted using 1–2 μg of total RNA using SuperScript III RT Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Quantitative PCR was performed using a SensiMix II Probe Kit (Bioline Reagents, London, United Kingdom) and the Universal ProbeLibrary (Roche Diagnostics) and analyzed using a CFX Connect Real-Time System and CFX Manager Software (Bio-Rad, Hercules, CA). Relative fold expression was normalized to β-actin (ACTB; TaqMan) and hydroxymethylbilane synthase (HMBS) sample controls using the primer sequences shown in Table 2.

Chan A.S., Clairfeuille T., Landao-Bassonga E., Kinna G., Ng P.Y., Loo L.S., Cheng T.S., Zheng M., Hong W., Teasdale R.D., Collins B.M, & Pavlos N.J. (2016). Sorting nexin 27 couples PTHR trafficking to retromer for signal regulation in osteoblasts during bone growth. Molecular Biology of the Cell, 27(8), 1367-1382.

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Quantitative Analysis of Endothelial and Hematopoietic Gene Expression

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Total RNA was purified from zebrafish endothelial cells, HSPCs, and HUVECs using the RNeasy Plus Mini kit (QIAGEN). Complementary DNA was reverse transcribed using the SuperScript III RT kit (Thermo Fisher Scientific) and a mixture of poly-A and random hexamer primers. Quantitative PCR was performed using SSOFast Supermix with EvaGreen (Bio-Rad Laboratories) and a Bio-Rad thermal cycler. The following primer sequences were used in the study: zf-18s: 5′-TCGCTAGTTGGCATCGTTTATG-3′ (forward), 5′-CGGAGGTTCGAAGACGATCA-3′ (reverse); zf-cxcl8: 5′-TCATTGAAGGAATGAGCTTGAGAG-3′ (forward), 5′-CCAGTTGTCATCAAGGTGGC-3′ (reverse); zf-cxcr1: 5′-GTGATCGTACGCGCTATGGA-3′ (forward), 5′-ATTCGGGTTGCTAATCGCCA-3′ (reverse) ; hs-CXCL12: 5′-ACATGGCTTTCGAAGAATCG-3′ (forward), 5′-GCTGGTCCTCGTGCTGAC-3′ (reverse); hs-survivin: 5′-TTGGTGAATTTTTGAAACTGGA-3′ (forward), 5′-CTTTCTCCGCAGTTTCCTCA-3′ (reverse); hs-VEGFa: 5′-AGGGCAGAATCATCACGAAGT-3′ (forward), 5′-AGGGTCTCGATTGGATGGCA-3′ (reverse) ; hs-CXCL8 5′-AAATTTGGGGTGGAAAGGTT-3′ (forward), 5′-TCCTGATTTCTGCAGCTCTGT-3′ (reverse).

Blaser B.W., Moore J.L., Hagedorn E.J., Li B., Riquelme R., Lichtig A., Yang S., Zhou Y., Tamplin O.J., Binder V, & Zon L.I. (2017). CXCR1 remodels the vascular niche to promote hematopoietic stem and progenitor cell engraftment. The Journal of Experimental Medicine, 214(4), 1011-1027.

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Priming Human PBMCs with AngII and LPS

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Human PBMCs were isolated from the blood of healthy volunteers using a standard Ficoll protocol. Subsequently, cells were treated with AngII (100uM) for 24 hours followed by LPS induction (100ng/ml) for an additional 24 hours. IL-10 (20ng/ml) or control treatment (PBS) was performed in combination with AngII priming and before LPS stimulation. Cells were harvested using Qiazol and RNA was isolated using the Qiagen miRNeasy kit. RNA was quantified by Nanodrop (Thermo Fisher Scientific). mRNA was reverse transcribed using the SuperScript III RT kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and expression levels were quantified with standard qRT-PCR (TaqMan) with the following assays: GAPDH (internal control), TNF, IL6, TGFB1, MRC1, CD163, FOXP3, GZMB, IL10 (all Thermo Fisher Scientific). Amplification was performed using a Quantstudio 12K Flex Real-Time PCR system (Thermo Fisher Scientific). Results were normalised to GAPDH.

Adam M., Kooreman N., Jagger A., Wagenhaeuser M.U., Mehrkens D., Wang Y., Kayama Y., Toyama K., Raaz U., Schellinger I.N., Maegdefessel L., Spin J.M., Hamming J.F., Quax P.H., Baldus S., Wu J.C, & Tsao P.S. (2018). Systemic upregulation of IL-10 using a non-immunogenic vector reduces growth and rate of dissecting abdominal aortic aneurysm. Arteriosclerosis, thrombosis, and vascular biology, 38(8), 1796-1805.

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Quantifying Gene Expression in Liver Tissue

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Total RNA was obtained from liver tissue with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocols, and cDNA for RT-qPCR was synthesized using oligo d(T) and a Superscript III RT kit (Thermo Fisher Scientific, Inc.). qPCR was conducted using a Quantitative SYBR-Green RT-PCR kit (Takara Bio, Inc., Otsu, Japan) and an Applied Biosystems 7500 system (Thermo Fisher Scientific, Inc.). All reactions were conducted in a 20 µl reaction volume in triplicate. The relative expression levels for a target gene were normalized to β-actin. Specificity was verified by melting curve analysis and agarose gel electrophoresis. FOXO3a, nuclear factor (NF)-κB and β-actin were obtained from Wuhan GuGe Biotechnology, (Wuhan China). Primers were as follows: FOXO3a sense, 5′-AACAGTACCGTGTTCGGACC-3′ and anti-sense, 5′-AGTGTCTGGTTGCCGTAGTG-3′; NF-κB sense, 5′-GCTCCTTTTCTCAAGCCGATGT-3′ and anti-sense, 5′-CGTAGGTCCTTTTGCGTTTTTC-3′; and β-actin sense, 5′-GTTACAGGAAGTCCCTCACCC-3′ and anti-sense, 5′-CAGACCTGGGCCATTCAGAAA-3′. Data were analyzed using the comparative Cq (2^−∆∆Cq) (20 (link)).

Xiao Q., Ye Q., Wang W., Xiao J., Fu B., Xia Z., Zhang X., Liu Z, & Zeng X. (2017). Mild hypothermia pretreatment protects against liver ischemia reperfusion injury via the PI3K/AKT/FOXO3a pathway. Molecular Medicine Reports, 16(5), 7520-7526.

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Temporal Expression of Nucleoporins

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Comparison of expression levels of nup transcripts in synchronized cells was performed by RT-qPCR. Briefly, cells were synchronized in S-phase and released; cells were then harvested every hour. Total RNA was isolated using the RNeasy Plus Mini kit (Qiagen) per the manufacturer’s protocol. Superscript III RT kit (Thermo Fisher Scientific) was used to synthesize cDNA using Oligo-dT primers (Thermo Fisher Scientific). qPCR of the cDNA template was performed by the iTaq Universal SYBR Green Supermix (Bio-Rad) with 0.2 pmol/µl of gene-specific primers (Integrated DNA Technologies; Table S4). A region of the GAPDH cDNA was amplified as an internal control. The specificity of each amplicon was verified by a melt curve analysis.

Vishnoi N., Dhanasekeran K., Chalfant M., Surovstev I., Khokha M.K, & Lusk C.P. (2020). Differential turnover of Nup188 controls its levels at centrosomes and role in centriole duplication. The Journal of Cell Biology, 219(3), e201906031.

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Quantitative RNA Expression Analysis

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48 hours post-treatment, RNA was harvested using TRIzol (Invitrogen) reagent according to manufacturer’s instructions. RNA concentration was quantified by Nanodrop (ThermoFisher).
Reverse transcription was performed using the SuperScript III RT kit (ThermoFisher) using 1 μg of input RNA. The cDNA samples were diluted 1:20 and 1μl of sample template was added to a reaction mix containing 2.5μl of SYBR^® green mix, 1.5μl of primer (5μM stock; Table 1) and 1μl of RNase-free water. The qPCR reaction was run using under the following conditions:

Chen K., Phan T., Lin A., Sardo L., Mele A.R., Nonnemacher M.R, & Klase Z. (2020). Morphine exposure exacerbates HIV-1 Tat driven changes to neuroinflammatory factors in cultured astrocytes. PLoS ONE, 15(3), e0230563.

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Cloning and Stable Expression of FLAG-tagged Proteins

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All primer sequences used for cloning are provided in Table S4. RIOK3 (NM_003831.4), as well as both long (NM_001300829) and short (NM_001280) isoforms of CIRBP, were cloned by PCR (HiFi PCR premix, Clontech) from cDNA from Huh7 cells prepared with the Superscript III RT kit (Thermo Fisher) using the oligo(dT)₂₀ primer. PCR products were inserted into pLEX-FLAG lentiviral vector between the NotI and XhoI sites using the InFusion HD cloning kit (Takara Bio) to generate constructs with N-terminal FLAG tags. Lentivirus was produced from 293T cells transfected with pLEX vectors and packaging plasmids psPAX2 and pMD2.G (provided by Duke Functional Genomics Facility). Huh7 cells were transduced by these lentiviruses and stable cell lines expressing FLAG-RIOK3, FLAG-CIRBP-S, and FLAG-CIRBP-L were selected using puromycin (2 μg/mL; Sigma-Aldrich). Single cell clones were obtained by serial dilution and verified by immunoblotting. Cell lines were maintained in cDMEM containing 1 μg/mL puromycin.

Gokhale N.S., McIntyre A.B., Mattocks M.D., Holley C.L., Lazear H.M., Mason C.E, & Horner S.M. (2019). Altered m6A Modification of Specific Cellular Transcripts Affects Flaviviridae Infection. Molecular cell, 77(3), 542-555.e8.

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Sequencing Recombinant T Cell Receptors

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For sequencing of recombinant TCRVs, RNA was extracted from virus stocks using the QIAamp Viral RNA Mini kit (Qiagen) according to the manufacturer’s instructions. Full-length genome cDNA was reverse transcribed with a primer binding to the conserved 3’ genome end using the SuperScript III RT kit (Thermo Fisher), also according to the manufacturer’s instructions. Subsequent amplification of individual PCR fragments for sequencing was accomplished with the iProof High Fidelity PCR kit (BioRad) using the primers listed in S2 Table. PCR products were cleaned up using the Macherey-Nagel NucleoSpin Gel and PCR Clean-up kit after which Sanger sequencing was performed using the same primers used for amplification, as well as the additional sequencing primers indicated in S2 Table. PCR products corresponding to the TCRV genome ends were amplified from cDNA by using 3′ and 5′ rapid amplification based on ligation-anchored PCR, as previously described [41 (link)].

Bohn P., Waßmann I., Wendt L., Leske A., Hoenen T., Tews B.A, & Groseth A. (2023). A dsRNA-binding mutant reveals only a minor role of exonuclease activity in interferon antagonism by the arenavirus nucleoprotein. PLOS Pathogens, 19(1), e1011049.

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Quantitative Analysis of Mechanosensitive Gene Expression

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Flies were allowed to lay eggs on molasses plates for 2.5 hours, so that most embryos collected were in nc14. Flies were either homozygous for the duplicated distal reporter on chromosome 2 or of the same genetic background (BDSC #9723) but did not have the transgene. The embryos collected from each plate were pooled and total RNA was extracted and purified using TRIzol (Thermo Fisher Scientific) and the Direct-zol RNA Miniprep kit (Zymo Research). cDNA was generated using SuperScript III RT Kit (Thermo Fisher Scientific). qPCR amplification was then done using the TaqMan Gene Expression Master Mix (Thermo Fisher Scientific). The data for each group, transgenic or non-transgenic, is from three separate biological replicates (i.e. the colored circles in Figure 4C are biological replicates), each done in technical triplicates. Relative RNA levels of each measured gene was calculated using the 2-ddC(t) method, using RpII140 as the reference gene. The TaqMan FAM probes used for each gene were DM01803576_g1 for Piezo, Dm01825813_g1 for Mcr, Dm01803642_g1 for Btk29A, and DM02134593_g1 for RpII140.

Waymack R., Gad M, & Wunderlich Z. (2021). Molecular competition can shape enhancer activity in the Drosophila embryo. iScience, 24(9), 103034.

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