Easyscript cdna synthesis supermix

Manufactured by Transgene

Sourced in China

EasyScript cDNA Synthesis SuperMix is a laboratory reagent used for the conversion of RNA into complementary DNA (cDNA). The SuperMix contains all the necessary components for efficient cDNA synthesis in a single, ready-to-use solution.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (5 mentions) Transscript mirna first strand cdna synthesis supermix, by Transgene (4 mentions) Transstart top green qpcr supermix, by Transgene (4 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Trizol reagent, by Merck Group (1 mentions) Transstart tip green qpcr supermix, by Transgene (1 mentions) Transstart top green qpcr supermix dye 2, by Transgene (1 mentions) Real time pcr detection system, by Bio-Rad (1 mentions) Universal sybr green supermix, by Bio-Rad (1 mentions) Rna extraction kit, by Omega Bio-Tek (1 mentions) Sybr premix ex taq 2, by Takara Bio (1 mentions) 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)

11 protocols using easyscript cdna synthesis supermix

Quantitative RT-PCR for miR-30 and Spastin mRNA

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TRIzol reagent (Invitrogen) was applied to total RNA extracts of HT22 or COS1 cells according to the manufacturer’s instructions. An EasyScript cDNA Synthesis SuperMix (TransGen Biotech, China) was used to reverse transcribe the mRNA. A TransScript^® miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech, China) was used for miRNA cDNA synthesis. The quantitative reverse transcription-polymerase chain reaction (PCR) was performed using TransStart Top Green quantitative PCR (qPCR) SuperMix (TransGen Biotech, China). miR-30 and mRNA expression levels were normalized to those of U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using a delta-delta-Ct method as described previously (Livak and Schmittgen, 2001 (link)). All PCR reactions were repeated at least three times. The primers specific to mature miR-30 were purchased from Qiagen. The following sense and anti-sense primers were used:
spastin (HT22): 5′-GAACCCGTCTTCTTTCTCGTC-3′, 5′-AATGGAGATGTACTCGAAGGC-3′;
spastin (COS1): 5′-AGCGGAACCTGTACTATTTCTC-3′, 5′-CTGCTTTCTCATCCTCATCGAT-3′;
MALAT1: 5′-CTCACTAAAGGCACCGAAGG-3′, 5′-GGCAGAGAAGTTGCTTGTGG-3′.

Jiang T., Cai Z., Ji Z., Zou J., Liang Z., Zhang G., Liang Y., Lin H, & Tan M. (2020). The lncRNA MALAT1/miR-30/Spastin Axis Regulates Hippocampal Neurite Outgrowth. Frontiers in Cellular Neuroscience, 14, 555747.

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Quantifying Extracellular Matrix Markers

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The expression levels of mRNAs encoding collagen I, tenascin C (TnC), collagen III, scleraxis (Scx), α-smooth muscle actin (α-SMA), and VEGFA were determined by real-time qPCR. Briefly, TRIzol reagent (Sigma) was applied to extract total RNA, reverse transcription was performed with EasyScript cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China), and real-time qPCR was performed with TransStart Top Green qPCR SuperMix (TransGen Biotech). Relative expression levels were determined using the 2^-ΔΔCt method, and GAPDH was used as the control. The primer sequences are shown in Table 1.

Xue Z., Chen Z., Wu T., Li R., Chen C., Liu J., Hou H., Zheng X, & Wang H. (2022). VEGFA-Enriched Exosomes from Tendon-Derived Stem Cells Facilitate Tenocyte Differentiation, Migration, and Transition to a Fibroblastic Phenotype. BioMed Research International, 2022, 8537959.

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Tissue-specific Transcriptome Analysis

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Total RNA was extracted from various tissues, including the midgut treated with 20E or RNAi, using TRIzol Reagent (Invitrogen). Total RNA was isolated from fat bodies of sixth-instar larvae at 72 and 96 h PE after either dsRNA injection or L. plantarum treatment. First-strand cDNA was synthesized using EasyScript cDNA synthesis SuperMix (TransGen Bio-tech). RT-qPCR analysis was performed using TransStart Tip Green qPCR SuperMix (TransGen Bio-tech). The housekeeping gene β-actin was used as an endogenous control. Primers used for RT-qPCR analysis are listed in Table S8 (Additional file 1).

Xiong P., Wang W.W., Liu X.S., Wang Y.F, & Wang J.L. (2024). A CTL − Lys immune function maintains insect metamorphosis by preventing gut bacterial dysbiosis and limiting opportunistic infections. BMC Biology, 22, 54.

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Quantifying Gene Expression in Insect Tissues

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Total RNA of fat bodies, PG, CA, and guts either from dsRNA injection or from E. mundtii injection were extracted using TRIzol (Invitrogen). After treatment with DNase I, first-strand cDNA was synthesized with 2 μg of total RNA using EasyScript cDNA synthesis SuperMix (TransGen Bio-tech). RT-qPCR was performed with TransStart Top Green qPCR SuperMix (TransGen Bio-tech). Each sample was assayed in triplicate, and the relative expression for each gene was calculated using the 2^−ΔCT method (ΔC_T = C_{T, tested gene} −C_{T, Haβ-actin}). Primers designed for RT-qPCR analysis are listed in S6 Table.

Wang W., Wang G., Zhuo X., Liu Y., Tang L., Liu X, & Wang J. (2020). C-type lectin-mediated microbial homeostasis is critical for Helicoverpa armigera larval growth and development. PLoS Pathogens, 16(9), e1008901.

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Quantitative Gene Expression Analysis

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Total RNA was extracted with TRIzol. EasyScript cDNA Synthesis SuperMix (Transgen, China) was used to prepare cDNA. Real-time PCR was conducted in triplicate with TransStart Top Green qPCR SuperMix (+Dye II) (TransGen). Supplementary Table 1 lists the primers used in this study. β-Actin and U6 were detected as internal controls. The relative amount of RNA was calculated by the 2^−ΔΔCt method.

Wang S., Yang X., Xie W., Fu S., Chen Q., Li Z., Zhang Z., Sun T., Gong B, & Ma M. (2021). LncRNA GAPLINC Promotes Renal Cell Cancer Tumorigenesis by Targeting the miR-135b-5p/CSF1 Axis. Frontiers in Oncology, 11, 718532.

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Transcription Analysis: RNA Extraction and qPCR

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Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. To reverse transcribe mRNA, 1 μg RNA was used to reverse-transcribe mRNA into cDNA with an EasyScript cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) was used. For miRNA cDNA synthesis, a TransScript miRNA First-Strand cDNA Synthesis SuperMix (TransGen) was used. RT-qPCR was performed using TransStart Top Green quantitative PCR SuperMix (TransGen). Primer sequences used are listed in Table 1. For quantification, genes were normalized to Gapdh and miRNA to U6.

Liu Y.J., Wang H.J., Xue Z.W., Cheang L.H., Tam M.S., Li R.W., Li J.R., Hou H.G, & Zheng X.F. (2021). Long noncoding RNA H19 accelerates tenogenic differentiation by modulating miR-140-5p/VEGFA signaling. European Journal of Histochemistry : EJH, 65(3), 3297.

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Cardiac Gene Expression Analysis

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Total RNA was extracted from cardiomyocytes or H9c2 cells using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. EasyScript cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) was used to synthesize cDNA from mRNA, and TransScript^® miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech) was used to synthesize cDNA from miRNA. Quantitative PCR (qPCR) was performed using TransStart Top Green qPCR SuperMix (TransGen Biotech). mRNA levels were normalized to that of actin, and miRNA levels were normalized to that of U6, using a delta-delta-Ct method as previously described.^{36 ,37 (link)} All experiments were independently performed in triplicate. The following primers were used: 5’-CTCACTAAAGGCACCGAAGG- 3’ and 5’-GGCAGAGAAGTTGCTTGTGG- 3’ (MALAT1); 5’-CTTCGGGGGTAGGATTGAC- 3’ and 5’-CTTGGGATCTTTTGCGATCT-3’ (ANP); 5’-TGATTCTGCTCCTGCTTTTCC-3’ and 5’-TTCTTTTGTAGGGCCTTGGTC- 3’ (BNP); 5’-CGCCTGTCAGCTTGTAAATG- 3’ and 5’-ACAACCCCTACGATTATGCG-3’ (β- MHC); 5’-TCCTGGTAGGCCAACAGGCT-3’ and 5’-AGCTAATGTTGAGCTGCACTTG- 3’ (HMGB2); 5’-CCGCATCTTCTTGTGCAGTG- 3’ and 5’-GAGAAGGCAGCCCTGGTAAC- 3’ (GAPDH); 5’-GGGAACATTCAACGCTGTCG-3’ and 5’-GTGCGTGTCGTGGAGTCG-3’ (miR-181a); and 5’- CTCGCTTCGGCAGCACA-3’ and 5’-AACGCTTCACGAATTTGCGT- 3’ (U6).

Chen F., Li W., Zhang D., Fu Y., Yuan W., Luo G., Liu F, & Luo J. (2022). MALAT1 regulates hypertrophy of cardiomyocytes by modulating the miR-181a/HMGB2 pathway. European Journal of Histochemistry : EJH, 66(3), 3426.

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Transcriptomic Analysis of Insect Midgut Development

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Total RNA was extracted from the midguts of sixth-instar larvae at 24 h PE (feeding; MgF) and 72 h PE (wandering; MgW), and those at 72 h PE pretreated with dsCD209 or dsGFP using TRIzol (Invitrogen, Carlsbad, CA, USA). The cDNA libraries (MgF-1, -2, -3, -4; MgW-1, -2, -3, -4; dsCD209-1, -2, -3; dsGFP-1, -2, and -3) were constructed as described [59 (link)]. Briefly, mRNAs were isolated using oligo(dT) beads and then fragmented. Double-stranded cDNAs were synthesized using EasyScript cDNA synthesis SuperMix (TransGen Bio-tech, Beijing, China), followed by sequencing on the Illumina Novaseq 6000 platform (San Diego, USA) at Majorbio Bio-Pharm Technology Co., Ltd. Clean reads were obtained after filtration of raw reads and aligned to the H. armigera genome (https://www.ncbi.nlm.nih.gov/genome/13316?genome_assembly_id=319039) using TopHat software (http://tophat.cbcb.umd.edu/). Mapped reads were assembled and all unigenes were run in BLASTx against the non-redundant database. DEGs in the libraries (MgW vs. MgF; dsCD209 vs. dsGFP) were determined using a fold cut-off change value ≥ 2 and adjusted p-value < 0.05.

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Validation of Differentially Expressed circRNAs in iCCA

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To verify the authenticity of the DE circRNAs that were identified from the iCCA, 7 DE circRNAs (hsa_circ_0006114, hsa_circ_0015839, hsa_circ_0058010, hsa_circ_0000020, hsa_circ_0001727, hsa_circ_0006633, and hsa_circ_0026920) were randomly selected for validation with the real-time PCR. According to the flanking sequences of the junction sites of these circRNAs, the divergent primers were designed to do the validation (Table 1). After extracting the total RNAs from the iCCA and iCCAP samples, as mentioned above, cDNA was obtained using the EasyScript cDNA Synthesis SuperMix (Transgen Biotech, Beijing, China). The expression levels of these DE circRNAs were detected using the real-time PCR in iCCA (n = 10) and iCCAP (n = 10). The real-time PCR was done using the Real-Time PCR Detection System (Bio-Rad, United States) with the Universal SYBR Green Supermix (Bio-Rad, United States). We used β-actin (internal standard control) as the reference gene to calculate the expression levels of the circRNAs using the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)). All the experiments were repeated in triplicate.

Liang Z., Liu L., Guo X., Wu X., Yu Y.L., Yu Z., Hu X., Zhang X, & Wang J. (2022). The expression profiles of circular RNAs and competing endogenous RNA networks in intrahepatic cholangiocarcinoma. Frontiers in Cell and Developmental Biology, 10, 942853.

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Characterization of GRDP2 T-DNA Mutants

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Three T-DNA mutant lines Sail_387_D04, SALK_412A10, and SALK_112794C for the GRDP2 (At4g37900) gene were acquired from the Arabidopsis Biological Resource Center¹. pDR5:GFP, pPIN1:PIN1-GFP, pPIN2:PIN2-GFP, pPIN7:PIN7-GFP, and pYUCCA5-GFP-GUS (from Dr. Xu Chen, Fujian Agriculture and Forestry University, Fuzhou, China). pSPL:GUS, pKNU:KNU-VENUS, pAKV:H2B-YFP, pDD45:GFP, and pDD65:GFP (from Dr. Yuan Qin, Fujian Agriculture and Forestry University, Fuzhou, China). The T-DNA insertion was identified by primers (Supplementary Table 1) obtained from the SIGnAL website². GRDP2 expression level in T-DNA mutant lines was confirmed by RT-qPCR using the primers listed in Supplementary Table 1.
Total RNA was extracted from inflorescence tissues using the RNA extraction Kit (Omega Bio-Tek, Shanghai, China) following the manufacturer’s protocol. EasyScript^® cDNA Synthesis SuperMix (Transgen, Beijing, China) was used for the cDNA preparation. The RT-qPCR was conducted using TransStart^® Top Green qPCR SuperMix (Transgen, Beijing, China). HK2 (AT5G35750) gene was used as a reference gene (Supplementary Table 1; Zhao et al., 2018 (link)). These assays were conducted for three biological replicates, and the results are shown as the mean ± standard deviations.

Wang L., Liu Y., Aslam M., Jakada B.H., Qin Y, & Cai H. (2021). The Glycine-Rich Domain Protein GRDP2 Regulates Ovule Development via the Auxin Pathway in Arabidopsis. Frontiers in Plant Science, 12, 698487.

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