Superscript double stranded cdna kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The SuperScript double-stranded cDNA kit is a laboratory tool designed to synthesize double-stranded complementary DNA (cDNA) from RNA samples. It provides a reliable and efficient method for generating cDNA for various downstream applications, such as gene expression analysis, cDNA library construction, and Next-Generation Sequencing.

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Lab products found in correlation

Iq syber green supermix, by Bio-Rad (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Phenol extraction, by Thermo Fisher Scientific (1 mentions) Beacon designer 8, by Premier Biosoft (1 mentions) Rneasy mini column, by Qiagen (1 mentions) Experion rna stdsens analysis kit, by Bio-Rad (1 mentions) Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Bioanalyzer, by Agilent Technologies (1 mentions) Rna 6000 nano kit, by Agilent Technologies (1 mentions) Nanodrop nd 2000, by Thermo Fisher Scientific (1 mentions) Truseq rna kit, by Illumina (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Novaseq 6000, by Illumina (1 mentions) Hiseq x ten, by Illumina (1 mentions) Truseq rna sample preparation kit, by Illumina (1 mentions) Iq5 multicolor real time pcr detection system, by Bio-Rad (1 mentions)

7 protocols using superscript double stranded cdna kit

Transcriptional Analysis of Transthyretin in Hsf/V30M Mice

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Liver and colon mRNA (n = 6 per group Hsf/V30M; n = 5 WT) was isolated using phenol extraction (Invitrogen, Carlsbad, CA, USA). Sciatic nerve from Hsf/V30M mice was dissected free from surrounding tissue (n = 5) and mRNA extraction performed using RNeasy Mini columns (Qiagen, Gaithersburg, MD, USA). cDNA was synthesized with the SuperScript double-stranded cDNA Kit (Invitrogen). The quality of extracted RNA was assessed with Experion RNA StdSens Analysis Kit (Bio-Rad, Hercules, CA, USA); qPCR was performed in duplicates using iQ Syber Green Super Mix (Bio-Rad) and reactions were run on an Bio-Rad iQ5 software.
Primer sequences were designed using Beacon Designer 8™ (Premier Biosoft, Palo Alto, CA, USA) for TTR (Forward: 5’-ATTCTTGGCAGGATGGCTTC-3’, Reverse: 5’-CAGAGGACACTTGGATTCACC-3’); Ttr (Forward: 5’-AGCCCTTTGCCTCTGGGAAGAC-3’, Reverse: 5’-TGCGATGGTGTAGTGGCGATGG-3’); glyceraldehyde 3-phosphate dehydrogenase (Gapdh) (Forward: 5’-GCCTTCCGTGTTCCTACC-3’, Reverse: 5’-AGAGTGGGAGTTGCTGTTG-3’) and 18S (Forward: 5’-AAATCAGTTATGGTTCCTTTGGTC-3’, Reverse: 5’-GCTCTAGAATTACCACAGTTATCCAA-3’). Differential expression was determined by the 2^-^∆∆CT method.

Gonçalves N.P., Costelha S, & Saraiva M.J. (2014). Glial cells in familial amyloidotic polyneuropathy. Acta Neuropathologica Communications, 2, 177.

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RNA Isolation and Gene Expression Analysis

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RNA samples from each cell line were purified using TRIZOL (Invitrogen), and double-stranded complementary DNA (cDNA) was generated using the SuperScript double-stranded cDNA kit (Invitrogen). Samples were then submitted to Roche NimbleGen for subsequent hybridization and downstream processing using the NimbleGen 12 × 135 k mouse gene expression array platform, which assays 44,170 target genes with three separate 60mer probes per transcript. Biological replicates were performed for all cell lines.

West J.A., Cook A., Alver B.H., Stadtfeld M., Deaton A.M., Hochedlinger K., Park P.J., Tolstorukov M.Y, & Kingston R.E. (2014). Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming. Nature Communications, 5, 4719.

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RNA Extraction and cDNA Synthesis

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Samples were directly transferred from −80 to a 65°C water bath for 15 min to lyse cells and mixed with 50 μl chloroform for 5 min at room temperature. The aqueous phase was recovered after centrifugation for 15 min at 13,000 g, 4°C. Total RNA was purified using RNeasy Mini Kit (Qiagen) with on-column DNase I treatment following the manufacturer's instructions, and eluted in 15 μl RNase-free water. RNA concentration was quantified on a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Inc.). Concentrations and ratios A_260/280 and A_260/230 are given in Supplementary Table 1. RNA integrity was confirmed by 0.8% agarose gel electrophoresis. The absence of genomic DNA was confirmed by PCR on purified RNA. The cDNAs were synthesized from 10 μg total RNA using the Superscript Double Stranded cDNA kit (Invitrogen), purified by phenol:chloroform:isoamylic alcohol extraction (v/v, 25:24:1), recovered by ethanol precipitation and resuspended in 15 μl nuclease-free water. The cDNA quality was confirmed on a Bioanalyzer (Agilent) using the RNA 6000 Nano kit (Supplementary Figure 1).

Thomas F., Bordron P., Eveillard D, & Michel G. (2017). Gene Expression Analysis of Zobellia galactanivorans during the Degradation of Algal Polysaccharides Reveals both Substrate-Specific and Shared Transcriptome-Wide Responses. Frontiers in Microbiology, 8, 1808.

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Transcriptome Analysis of Gut Tissue Samples

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Gut tissue samples from the CTR and treatment groups (4 replicates) were collected for total RNA extraction using the TRIzol reagent (Invitrogen, Thermo Fisher Scientific, USA). Genomic DNA was removed using DNases I (Takara, Japan) and the RNA quality and integrity were measured using the ND-2000 NanoDrop (Thermo Fisher Scientific, USA) and agarose gel electrophoresis (1%). Total RNA (1 μg) was used for the construction of transcriptome libraries using the TruSeq^TM RNA kit (Illumina, CA, USA). The mRNA was isolated with oligo (dT) beads according to the polyA selection method. Double-stranded cDNA was synthesized using the SuperScript double-stranded cDNA kit (Invitrogen, Thermo Fisher Scientific, USA) and was subjected to end repair. Phosphorylation, and ‘A’ base addition. Paired-end RNA-Seq library (2 × 150 bp) was sequenced with Illumina NovaSeq 6000 (CA, USA). Furthermore, the reads quality CTR, transcript assembling, expression analysis, functional annotation, identification of differentially expressed genes, and function enrichments were performed as previously described [35] , [36] (link).

Muhammad A., Zhang N., He J., Shen X., Zhu X., Xiao J., Qian Z., Sun C, & Shao Y. (2023). Multiomics analysis reveals the molecular basis for increased body weight in silkworms (Bombyx mori) exposed to environmental concentrations of polystyrene micro- and nanoplastics. Journal of Advanced Research, 57, 43-57.

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Transcriptional Analysis of EPS Biosynthesis

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Transcriptional analysis was performed on genes involved in EPS biosynthesis in S. thermophilus IMAU20561 grown for 5 h and 10 h in M17 medium containing different nitrogen sources, such as either soy peptone, tryptone, casein peptone as the sole sources or the complex nitrogen source typical of the M17 medium. The construction of the transcriptome library was done by the Shanghai Meiji Biological Analysis and Testing Co., Ltd. (Meiji, Shanghai) with the TruSeqTM RNA sample preparation Kit (Illumina, San Diego, CA). The mRNA was fragmented using metal ions and double-stranded cDNA was reverse transcribed with random primers using the SuperScript double-stranded cDNA kit (Invitrogen, CA). The second cDNA strand was synthesized, with dUTP instead of deoxythymidine triphosphate (dTTP), cDNA ends were patched with End Repair Mix and phosphorylated at the 5′ end and adenylated at the 3′ end. The cDNA library-enriched and the PCR were amplified using Phusion DNA polymerase (NEB). RNA-seq sequencing was done using Illumina HiSeq X Ten (2 × 150 bp).

Wang Y., Peng Q., Liu Y., Wu N., He Y., Cui X, & Dan T. (2024). Genomic and transcriptomic analysis of genes involved in exopolysaccharide biosynthesis by Streptococcus thermophilus IMAU20561 grown on different sources of nitrogen. Frontiers in Microbiology, 14, 1328824.

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Mouse Gene Expression Profiling

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RNA samples from each cell line were purified using TRIZOL (Invitrogen), and double-stranded cDNA were generated using the SuperScript double-stranded cDNA kit (Invitrogen). Samples were then submitted to Roche NimbleGen for subsequent hybridization and downstream processing using the NimbleGen 12×135k mouse gene expression array platform which assays 44,170 target genes with 3 separate 60mer probes per transcript. Biological replicates were performed for all cell lines.

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Quantitative RT-PCR Analysis of Mouse Genes

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First-strand cDNA was synthesized with the SuperScript double-stranded cDNA Kit (Invitrogen, Carlsbad, CA), according to the manufacturers' instructions. Primers for target mouse genes were designed with the Beacon Designer software version 8 (Premier Biosoft International, Palo Alto, CA), and their sequences are displayed in Table 1. A standard calibration curve was performed by 10-fold serial cDNA dilutions to assess primer efficiency.
Real-time quantitative PCR was conducted in duplicates with the use of the iQ Syber Green Super Mix (Bio-Rad), and reactions were analyzed in the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). Melting curves were performed to detect nonspecific amplification products. Five biological replicates were used per group. Differential expression was determined by the 2 ÀDDC T method, using Gapdh as the housekeeping control gene.

Gonçalves N.P., Martins D, & Saraiva M.J. (2016). Overexpression of Protocadherin-10 in Transthyretin-Related Familial Amyloidotic Polyneuropathy. The American journal of pathology, 186(7).

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