Superscript 4 cellsdirect cdna synthesis kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The SuperScript™ IV CellsDirect™ cDNA Synthesis Kit is a laboratory product designed for the reverse transcription of cellular RNA into cDNA. It enables the conversion of RNA extracted directly from cells into complementary DNA for further analysis.

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CDNA synthesis kit (1264 citations) SuperScript IV (708 citations) SuperScript IV kit (115 citations) SuperScript cDNA synthesis kit (97 citations) SuperScript IV cDNA synthesis kit (28 citations) CellsDirect kit (7 citations) Superscript IV synthesis kit (4 citations) CDNA kits (3 citations) SuperScript IV cDNA Kit (2 citations) CDNA-IV kit (2 citations)

12 protocols using superscript 4 cellsdirect cdna synthesis kit

RNA Extraction and cDNA Synthesis Protocols

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For in vitro free uptake experiments, the total RNA was extracted using the NucleoSpin RNA XS (Macherey Nagel, Hoerdt, France) according to the manufacturer’s recommendations. For transfection experiments the SuperScript™ IV CellsDirect™ cDNA Synthesis kit (Thermo Fisher Scientific) was used according to manufacturer’s recommendations. For in vivo experiments, mouse organs were crushed in 2 mL tubes pre-filled with ceramic mixture in Precellys^®Cryolys^® Evolution (Bertin, Montigny le Bretonneux, France) with a QIAZOL lysis buffer (Qiagen, Venlo, The Netherlands). A volume of 150 µL of chloroform (Sigma Aldrich) was added to 750 µL of tissue homogenate and a phenol/chloroform separation was performed using centrifugation for 15 min at 6000× g at 4 °C. Aqueous phases were recovered and RNA extraction was performed with the RNeasy 96 QIAcube HT Kit (Qiagen) according to the manufacturer’s recommendations, in a QIACUBE HT instrument (Qiagen). Quality and quantity of total RNA was determined with DNF-471 RNA Kit-15 nt (Agilent, Santa Clara, CA, USA) in a Fragment Analyzer 5300 (Agilent). For reverse transcription (RT), 500 ng of total RNA were used (except for cells treated with SuperScript™ IV CellsDirect™ cDNA Synthesis), and cDNA synthesis was performed using the High-Capacity RNA-to-cDNA™ kit (Thermo Fisher Scientific) according to the manufacturer’s recommendations.

Broc B., Varini K., Sonnette R., Pecqueux B., Benoist F., Masse M., Mechioukhi Y., Ferracci G., Temsamani J., Khrestchatisky M., Jacquot G, & Lécorché P. (2024). LDLR-Mediated Targeting and Productive Uptake of siRNA-Peptide Ligand Conjugates In Vitro and In Vivo. Pharmaceutics, 16(4), 548.

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FACS Sorting and qPCR Analysis of HSC Markers

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To detect TPX2, CDCA8, PCNA, MLLT3 and TYMS FACS sorted human BM VAP-1⁺ and VAP-1⁻ HSC (Lin⁻CD34⁺CD38⁻ CD45RA⁻ CD90⁺) were used. SuperScript IV CellsDirect cDNA Synthesis Kit (ThermoFisher Scientific) was applied for direct cDNA synthesis using sorted cell lysis protocol. One cell equivalent cDNA was used for RT-PCR using TaqMan Fast Advanced Master Mix (ThermoFisher Scientific). The following TaqMan Assays were used: Hs00187842_m1 (B2M), Hs00201616_m1 (TPX2), Hs00983655_m1 (CDCA8), Hs00427214_g1 (PCNA), Hs00426586_m1(TYMS) all from ThermoFisher Scientific. cDNA was analyzed by real-time qPCR (7900HT Fast Real-Time PCR System, Applied Biosystems). The results were analyzed in Applied Biosystems qPCR analysis modules. The expression values were normalized using β2 microglobulin expression as endogenous controls.

Iftakhar-e-Khuda I., Pessia A., Zheng S., Kankainen M., Kontro M., Karikoski M., Laurila J., Gerke H., Tadayon S., Hollmén M., Tang J., Imhof B.A., Salmi M, & Jalkanen S. (2021). Vascular adhesion protein-1 defines a unique subpopulation of human hematopoietic stem cells and regulates their proliferation. Cellular and Molecular Life Sciences, 78(23), 7851-7872.

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Monoclonal Antibody-based Protein Analysis

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Rabbit anti-mouse TRIAP1 monoclonal antibody (ab182858), rabbit anti-mouse GAPDH monoclonal antibody (ab8245), horseradish peroxidase labeled immunoglobulin conjugate-rabbit anti-mouse IgG conjugate (ab233006) were purchased from Abcam (United Kingdom); SuperScript IV Reverse Transcriptase (M614272), SuperScript IV One-Step RT-PCR Kit (M33253), SuperScript IV CellsDirect cDNA Synthesis Kit (M39128), RNase Inhibitor (M712192), TRIZOL (M33253), cMTT (M2003), Annexin V-FITC apoptosis detection kit (M2118), MEM (M8042), RPMI-1640 medium (R8758), FBS (F8687) were purchased from Thermo Fisher Scientific. CO₂ incubator (Shanghai Boxun, cat: BC-J160S), 4°C centrifuge (eppendorf, centrifuge 5415R), PCR machine (eppendorfrealplex), ultra-clean workbench (Shanghai Boxun model SW-CJ-2FD), inverted fluorescence electron microscope (Leica DMI3000B), electrophoresis instrument (Tanon EPS300), GloMax^® Discover microplate reader (Promega, United States), Olympus inverted microscope GX41 (Olympus, Japan), CytoFLEX_ flow cytometer (Baker Mancourt, United States).

Yan J., Dai L., Yuan J., Pang M., Wang Y., Lin L., Shi Y., Wu F., Nie R., Chen Q, & Wang L. (2022). miR-107 Inhibits the Proliferation of Gastric Cancer Cells In vivo and In vitro by Targeting TRIAP1. Frontiers in Genetics, 13, 855355.

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Quantitative Real-Time PCR Analysis

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Three replicates for each sample were for qRT-PCR. cDNA was synthesized using a SuperScript IV CellsDirect cDNA Synthesis Kit (ThermoFisher, United States) according to the manufacturer’s instructions. qRT-PCR was performed on a LightCycler 480 Real-time PCR (Roche Life Science, Germany) with the SYBR Green Master Mix (ThermoFisher, United States) according to the manual. Normalization of all genes was performed with TUBB as an internal control. The primers for each gene are listed in Supplementary Table S4. The expression level for each gene was calculated from the cycle threshold using the 2^−ΔΔCT method.

Li J., Gao X., Chen X., Fan Z., Zhang Y., Wang Z., Shi J., Wang C., Zhang H., Wang L, & Zhao Q. (2023). Comparative transcriptome responses of leaf and root tissues to salt stress in wheat strains with different salinity tolerances. Frontiers in Genetics, 14, 1015599.

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Quantification of ACTG2 Gene Expression

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The total RNA of the tested cells extracted by TRlzol (ThermoFisher Scientific, USA) was reverse transcribed to cDNA (SuperScript IV CellsDirect cDNA Synthesis Kit, ThermoFisher Scientific, USA), after which 20 μg were taken for amplification under the reaction conditions of 95 ℃ for the 30 s, 95 ℃ for 5 s, 65 ℃ for 30 s and 72 ℃ for 30 s, for 40 cycles. ACTG2 expression by PCR instrument (Preflex PCR instrument, ThermoFisher Scientific, USA), normalized against GAPDH, was computed by 2 -ΔΔCT . The primer sequences were designed and synthesized by Su-zhou GENEWIZ Biotechnology Co.

Liu H., Xiang P., Miao W., Liu H., Shen H, & Xue S. (2023). Analysis of the Regulatory Effect of ACTG2 on Biological Behavior of Bladder Cancer Cells Based on Database Screening. Cellular and molecular biology (Noisy-le-Grand, France), 69(1).

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Quantifying MTP Expression in Huh-7 Cells

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The total RNA from control and MTP KO Huh-7 cells was isolated using TRIzol reagent (ThermoFisher Scientific, # 15596018) according to manufacturer’s protocol. The RNA quality and quantity was assessed by Nanodrop (ThermoFisher Scientific, # ND-ONE-W). The cDNA was synthesized using random primers and 2 μg of total RNA using SuperScript IV CellsDirect cDNA Synthesis Kit (Invitrogen, # 11750150) as per manufacturer’s protocol. For RNA controls, the same amount of RNA was used without reverse transcriptase. Expressions of MTP and reference gene 18S in control and MTP-KO cells were analyzed by quantitative real time PCR (qPCR). The qPCR was performed using appropriately diluted cDNA and RNA controls using 2× qPCR kit (Eurogentec, #RT-SN10-05) in Real time PCR machine QuantStudio3 (Applied Biosystems, #A28131).

Anaganti N., Chattopadhyay A., Poirier J.T, & Hussain M.M. (2022). Generation of hepatoma cell lines deficient in microsomal triglyceride transfer protein. Journal of Lipid Research, 63(9), 100257.

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RNA Extraction and qPCR Analysis

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For cells, total RNA was isolated from 1 × 10⁷ cells by AllPrep® DNA/RNA Micro Kit (Qiagen, Shanghai, China) according to the manufacturer's instructions. cDNA was synthesized with Transcript All‐in‐one First‐Strand cDNA Kit (TransGen, China). For embryo samples, total RNA was extracted from 200 blastomeres and complementary DNA (cDNA) was synthesized using the SuperScript™ IV CellsDirect™ cDNA Synthesis kit (Invitrogen, USA). This process adhered to the instructions provided by the manufacturer. StepOnePlus™ Real‐Time PCR system (Applied Biosystems) was used for qPCR assay. The primer sequences for qPCR are listed in Table S1. The mRNA level was analysed by the 2^−ΔΔCt method.

Zhang M., Zhai Y., An X., Li Q., Zhang D., Zhou Y., Zhang S., Dai X, & Li Z. (2023). DNA methylation regulates RNA m6A modification through transcription factor SP1 during the development of porcine somatic cell nuclear transfer embryos. Cell Proliferation, 57(5), e13581.

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Total RNA Extraction and cDNA Synthesis for Oocytes and Ovary

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Total RNA was exacted from 50 oocytes and reversed to cDNA using SuperScript™ IV CellsDirect™ cDNA Synthesis Kit (Invitrogen). The total RNA of ovary was extracted using TRIzol (Takara, Japan) and reversed to cDNA using RevertAid Master Mix (https://www.thermofisher.cn/document-connect/document-connect.html?url=https://assets.thermofisher.cn/TFS-Assets/LSG/manuals/MAN0018936_RevertAidMasterMix_UG.pdf; Thermo Fisher Scientific). Real‐time polymerase chain reaction (RT‐PCR) was conducted using PowerUp SYBR Green Master Mix (https://www.thermofisher.cn/document-connect/document-connect.html?url=https://assets.thermofisher.cn/TFS-Assets/LSG/manuals/100031508_PowerUp_SYBRgreen_QRC.pdf; Applied Biosystems). The primers used for quantitative RT PCR were listed in Table 1.

Wang L., Chen Y., Wei J., Guo F., Li L., Han Z., Wang Z., Zhu H., Zhang X., Li Z, & Dai X. (2022). Administration of nicotinamide mononucleotide improves oocyte quality of obese mice. Cell Proliferation, 55(11), e13303.

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Quantifying Gene Expression in Embryos

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The cloned and in vitro-fertilized embryos underwent 1st cDNA synthesis using the SuperScript™ IV CellsDirect™ cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Briefly, 8-cell embryos were lysed using SuperScript™ IV CellsDirect™ Lysis Solution, Lysis Enhancer, and DNase I. Lysis was terminated by the addition of SuperScript™ IV CellsDirect™ Stop Solution. SuperScript™ IV RT Master Mix was used for 1st cDNA synthesis at 25 °C for 10 min, 50 °C for 10 min, and 85 °C for 5 min. RT-qPCR was performed using Bio-Red CFX 96 at 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 32 s. The relative expression was calculated using the 2^−ΔΔCt method, with β-actin serving as the internal reference gene (Table 1).

Li W., Liu Y., Zhou G., Li Z., Wang Z., Wang L., Ma X, & Wang X. (2024). Comparison of Umbilical Cord Mesenchymal Stem Cells and Fibroblasts as Donor Nuclei for Handmade Cloning in Sheep Using a Single-Cell Transcriptome. Animals : an Open Access Journal from MDPI, 14(4), 589.

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Quantitative Real-Time PCR Analysis of Oocyte Genes

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In vitro maturated MII stage oocytes were used for qRT-PCR analysis. Total RNA was isolated from 50 oocytes and reversed to cDNA using SuperScript™ IV CellsDirect™ cDNA Synthesis Kit (Invitrogen, USA). The cDNA was then quantified by PowerUp SYBR Green Master Mix (Applied Biosystems, USA) using StepOnePlus™ Real-Time PCR System (Applied Biosystems, USA). The primer sequences were listed in Table 1.Table 1

Primer sequences of genes for quantitative real-time PCR.

Gene	Primer sequence
Sirt1	forward, 5’-ATGACGCTGTGGCAGATTGTT-3’
	reverse, 5’-CCGCAAGGCGAGCATAGAT-3’
Sirt3	forward, 5’-CTACATGCACGGTCTGTCGAA-3’
	reverse, 5’-GCCAAAGCGAAGTCAGCCATA-3’
Drp1	forward, 5’-CAGGTGGTGGGATTGGAGAC-3’
	reverse, 5’-CTGGCATAATTGGAATTGGTTT-3’
Opa1	forward, 5’-CCGAGGATAGCTTGAGGGTT-3’
	reverse, 5’-CGTTCTTGGTTTCGTTGTGA-3’
Mfn2	forward, 5’-TTCTTGTGGTCGGAGGAGTG-3’
	reverse, 5’-CTTTGGTGGTCCAGGTCAGT-3’
Sod1	forward, 5’-AACCAGTTGTGTTGTCAGGAC-3’
	reverse, 5’-CCACCATGTTTCTTAGAGTGAGG-3’
actin	forward, 5’-TTCAACACCCCAGCCATG-3’
	reverse, 5’-CCTCGTAGATGGGCACAGT-3’

Guo F., Wang L., Chen Y., Zhu H., Dai X, & Zhang X. (2024). Nicotinamide Mononucleotide improves oocyte maturation of mice with type 1 diabetes. Nutrition & Diabetes, 14, 23.

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