Cdna synthesis kit

Manufactured by Tiangen Biotech

Sourced in China

The cDNA Synthesis Kit is a laboratory equipment designed for the reverse transcription of RNA into complementary DNA (cDNA). The kit contains the necessary reagents and enzymes to efficiently synthesize cDNA from total RNA or mRNA samples.

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First-strand cDNA synthesis kit RT-PCR miRcute miRNA First-Strand cDNA Synthesis Kit Quant cDNA Synthesis Kit TIANScript II cDNA First Chain Synthesis kit TIANScript First Strand cDNA Synthesis Kit Quant cDNA first strand synthesis kit MiRcute First-Strand cDNA Synthesis Kit MiRcute miR First-Strand cDNA synthesis kit LnRcute lncRNA First-Strand cDNA Synthesis Kit TIANGEN Fastking cDNA First Strand Synthesis Kit

50 protocols using cdna synthesis kit

Quantitative Real-Time PCR Analysis

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The mycelia were harvested by shaking cultivation in YES broth, 28 °C, 180 rpm for 3 days. Total RNA was extracted from wild-type and mutant strains according to the instructions of RNApure Total RNA Kit (Aidlab Biotechnologies Co., Ltd., Beijing, China), and the RNA quality was checked by agarose gel and Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). The removal of gDNA and synthesized first-strand cDNA was performed using the cDNA synthesis Kit (TIANGEN, Beijing, China).
The cDNA template was diluted to 100 ng/μL by a trace nucleic acid analyzer, and the reaction system was prepared according to the instructions for use of the Power SYBR Green Master Mix Kit (TIANGEN, Beijing, China). Using the QuantStudio 6 Flex (Applied Biosystems, Carlsbad, CA, USA) qPCR system, the actin gene was used as a reference to normalize the target gene, and gene expression was calculated via the 2^−ΔΔCt method. The primers used for qPCR analysis are listed in Table S2. Three independent biological replicates were produced for this study.

Ma X., Jiang Y., Ma L., Luo S., Du H., Li X, & Xing F. (2022). Corepressors SsnF and RcoA Regulate Development and Aflatoxin B1 Biosynthesis in Aspergillus flavus NRRL 3357. Toxins, 14(3), 174.

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Carotid Artery Injury Transcriptome

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Three weeks after the artery injury, the mice were sacrificed and perfused with cold saline for 5 min. The injured carotid artery was then rapidly excised and stored with RNA stabilization solution (AM7021, ThermoFisher, Waltham, USA) at -20℃. Three to four carotid arteries were pooled per sample for total RNA isolation using TRIzol reagent (Tiangen Biotech, Beijing, China). RNA was reverse transcribed into complementary DNA using a cDNA synthesis kit (KR118, Tiangen Biotech) according to the manufacturer’s instructions.

Peng S., Wu W.Q., Li L.Y., Shi Y.C., Lin S, & Song Z.Y. (2023). Deficiency of neuropeptide Y attenuates neointima formation after vascular injury in mice. BMC Cardiovascular Disorders, 23, 239.

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Differential Expression Analysis and qPCR Validation

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Differential expression analysis was performed using the DESeq R package (1.18.0). DESeq provides statistical routines to determine differential gene expression using a model based on a negative binomial distribution. The differentially expressed genes were identified as described by Audic and Claverie (1997) . The original P values were corrected for multiple testing by following the false discovery rate procedure, and genes with adjusted P values below 0.05 were considered significant. Real-time qPCR.
RT-PCR was performed using a cDNA synthesis kit (Tiangen), according to the manufacturer's instructions. Specific RT-PCR primers were used to amplify 100-to 300-bp internal fragments of different genes. Real-time qPCR was performed using PowerUp SYBR green master mix (Invitrogen), according to the manufacturer's instructions on a 7300plus PCR system (Thermo Scientific). The 16S ribosomal RNA housekeeping gene was used to normalize prokaryotic gene expression values, and actin (ACT8) was used to normalize eukaryotic gene expression values. The relative expression levels of the target genes were calculated using the quantitation-comparative cycle threshold (DDCT) method.

Zhang C., Lv M., Yin W., Dong T., Chang C., Miao Y., Jia Y, & Deng Y. (2019). Xanthomonas campestris Promotes Diffusible Signal Factor Biosynthesis and Pathogenicity by Utilizing Glucose and Sucrose from Host Plants. Molecular plant-microbe interactions : MPMI, 32(2).

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Transcriptomic Analysis of Tunicamycin Stress in Algae

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RNA was extracted from algal cells on the 24th hour of tunicamycin stress using the RNAprep pure Plant Kit [Tiangen Biotech (Beijing) Co., Ltd., Beijing, China] and measured using NanoDrop 2000 (Thermo). RNA integrity was analyzed using the RNANano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, United States). cDNA was synthesized using random primers [cDNA synthesis kit, Tiangen Biotech (Beijing) Co., Ltd.]. NEB Next Ultra TMRNA Library Prep Kit for Illumina (NEB, United States) was used to generate sequence libraries. Sequence reads are available on the NCBI sequence read archives [GSE209809].
The library construction of transcriptomic and bioinformatic analysis were conducted as described in our previous paper [31 (link)]. Genes with |fold change|≥ 1.5 and FDR < 0.01 (adjusted P-value, determined by the Benjamini and Hochberg multiple-testing correction implemented in the ‘p. adjust’ method of R) were defined as differentially expressed genes. The value of FPKM was the average of three biological replicates.

Chen J., Du H., Liu Z., Li T., Du H., Wang W., Aslam M., Chen W., Li P., Luo H., Fang H, & Liu X. (2022). Endoplasmic reticulum-quality control pathway and endoplasmic reticulum-associated degradation mechanism regulate the N-glycoproteins and N-glycan structures in the diatom Phaeodactylum tricornutum. Microbial Cell Factories, 21, 219.

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Soybean Stress Response qPCR Analysis

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Soybean variety “Williams 82” was used for quantitative PCR (qPCR) analysis. Soybean seeds were sown into flowerpots and after 2 weeks of growth, soybean seedlings were irrigated with 200 mM NaCl and 250 mM mannitol, respectively. A total of 0.1 g of soybean leaf blade tissue was collected at 0, 2, 4, 6, 8, 10, and 12 h after the 200 mM NaCl induced salt stress and 250 mM mannitol induced drought stress treatments, and total RNA was extracted using an RNA extraction kit (ZOMANBIO, ZP405, China). Double stranded cDNA was obtained using a cDNA synthesis kit (TIANGEN, KR118, China) (Le et al., 2011 (link)). qPCR reactions were performed on the ABI Prism 7500 real-time PCR system (Applied Biosystems, Foster City, CA, United States); the Actin (100500082) gene was used as the internal control (Jian et al., 2008 (link)). The expression levels of nine GmPP2A-B′′ genes were determined using the 2^–Δ^Δ^CT method. All primers are listed in Supplementary Table 2.

Xiong Y., Fan X.H., Wang Q., Yin Z.G., Sheng X.W., Chen J., Zhou Y.B., Chen M., Ma Y.Z., Ma J, & Xu Z.S. (2022). Genomic Analysis of Soybean PP2A-B Family and Its Effects on Drought and Salt Tolerance. Frontiers in Plant Science, 12, 784038.

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Trigeminal Sensory Receptor mRNA Expression

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The mRNA levels of ZBTB20, TRPA1, TRPV1, and TRPM8 were analyzed by RT-PCR. The mice were decapitated, and the bilateral TGs were collected with sterilized instruments 30 min after histamine and CQ administration into bilateral cheeks. Total RNA was extracted with an RNA Extraction Kit (Takara). Isolated RNA was reverse-transcribed to synthesize first strand cDNA using a cDNA synthesis kit (Tiangen). The ABI 7500 Real-Time PCR System and SYBR Green I (Tiangen) were used for PCR. Real-time PCR mixtures were prepared, and the reaction conditions were set following the kit instructions. GAPDH was served as an internal control. The melting curve was used to evaluate the reliability of the PCR results. The threshold cycle (CT) value (the inflection point of the amplification curve) was determined, and the relative expression of target genes was calculated using the 2^−ΔΔCt method. The primer sequences for ZBTB20, TRPV1, TRPA1, TRPM8, and GAPDH are shown in Table 1.

Jia X., Dai M.H., Ren A.J., Wang T.T., Zhang W.J, & Zhang L. (2021). ZBTB20 in Nociceptive Neurons of the Trigeminal Ganglia Regulates Pruritus. Frontiers in Medicine, 8, 626554.

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Quantitative RT-PCR for Gene Expression Analysis

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Total RNA was isolated with the RNAprep Pure Cell/Bacteria Kit (TIANGEN Biotech, Beijing, China). First-strand cDNA synthesis was performed with a cDNA synthesis kit (TIANGEN Biotech). The levels of mRNAs were measured by quantitative RT-PCR (7900 HT; Applied Biosystems, Foster City, CA, USA) with SYBR Green Master Mix (TaKaRa Biotech, Dalian, China) and normalized to the level of β-actin mRNA. Sequences of forward and reverse primer pairs are as follows:
AUF1: forward 5′-CAGCAGAGTGGTTATGGGAAAGTATCC-3′ and reverse
5′-GACAGGAGCCACCTTCAAATGAATC-3′
β-actin: forward 5′-CCACGAGCGGTTCCGATG-3′ and reverse
5′-GCCACAGGATTCCATACCCA-3′
CCL2: forward 5′-TCTCTCTTCCTCCACCACCATG-3′ and reverse
5′-GCGTTAACTGCATCTGGCTGA-3′
CCL5: forward 5′-TTTCTACACCAGCAGCAAGTGC-3′ and reverse
5′-CCTTCGTGTGACAAACACGAC-3′
CXCL9: forward 5′-AGTGTGGAGTTCGAGGAACCCT-3′ and reverse
5′-TGCAGGAGCATCGTGCATT-3′
CXCL10: forward 5′-TAGCTCAGGCTCGTCAGTTCT-3′ and reverse
5′-GATGGTGGTTAAGTTCGTGCT-3′.
IL-6: forward 5′-GAGGATACCACTCCCAACAGACC-3′ and reverse
5′-AAGTGCATCATCGTTGTTCATACA-3′
IL-17RA: forward 5′-AGTGTTTCCTCTACCCAGCAC-3′ and reverse
5′-GAAAACCGCCACCGCTTAC-3′
IL-17RC: forward 5′-GCTGCCTGATGGTGACAATGT-3′ and reverse
5′-TGGACGCAGGTACAGTAAGAAG-3′
iNOS: forward 5′-CAGCTGGGCTGTACAAACCTT-3′ and reverse
5′-CATTGGAAGTGAAGCGTTTCG-3′.

Han X., Yang Q., Lin L., Xu C., Zheng C., Chen X., Han Y., Li M., Cao W., Cao K., Chen Q., Xu G., Zhang Y., Zhang J., Schneider R.J., Qian Y., Wang Y., Brewer G, & Shi Y. (2014). Interleukin-17 enhances immunosuppression by mesenchymal stem cells. Cell Death and Differentiation, 21(11), 1758-1768.

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Quantitative RT-PCR for Gene Expression Analysis

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The same RNA samples were reserve-transcribed into cDNA using a cDNA synthesis kit (Tiangen Biotech, Beijing, China) according to the manufacturer's protocol. qRT-PCR was performed in 96-well plates using a Roche LC480. Reactions (20 μL) were mixed with 10 μL of 2×SYBR SuperReal PreMix (Tiangen Biotech, Beijing, China), 2 μL of cDNA, 0.6 μL of forward primer, 0.6 μL of reverse primer and 6.8 μL of ddH₂O. The amplification procedure was as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. PCR primers are listed in Table 1. β-actin was amplified as an endogenous control. The relative mRNA level of each gene was calculated using the 2 ^−ΔΔCT method. At least three replicates were performed for each cDNA sample, and reactions were prepared in triplicate.

Wang C.M., Pan Y.Y., Liu M.H., Cheng B.H., Bai B, & Chen J. (2017). RNA-seq expression profiling of rat MCAO model following reperfusion Orexin-A. Oncotarget, 8(68), 113066-113081.

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Molecular Mechanisms of Irisin-Induced Macrophage Apoptosis

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Mouse RAW264.7 macrophages were purchased from Shanghai Institute of Biochemistry and Cell Biology (China). Irisin was bought from Phoenix Pharmaceuticals (USA). MTT, oil red O, tunicamycin (TM) and rabbit anti-β-actin monoclonal antibody were obtained from Sigma (USA). Rabbit antibodies against CHOP, Bcl-2, p-PERK and p-eIF2α were provided by Santa Cruz (USA). Rabbit anti-ATF6 polyclonal antibody was purchased from Abcam (USA). Goat anti-rabbit IgG and ready-to-use SABC-Cy3 immunohistochemical kit were bought from Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd. (China) and Wuhan Boster Biological Technology Co., Ltd. (China) respectively. DMEM high-glucose culture medium and fetal bovine serum were provided by Gibco (USA). RIPA lysis solution and BCA protein quantification kit were purchased from Solarbio (USA). Annexin V-FITC apoptosis detection kit was bought from Nanjing Keygen Biotech. Co., Ltd. (China). Trizol reagent was obtained from Invitrogen (USA). cDNA synthesis kit and Real Master Mix (SYBR Green) kit were provided by Tiangen Biotech (Beijing) Co., Ltd. (China). ECL kit and PVDF membrane were purchased from Pierce (USA) and Millpore (USA) respectively. Other reagents were all analytically pure.

Zheng G., Li H., Zhang T., Yang L., Yao S., Chen S., Zheng M., Zhao Q, & Tian H. (2017). Irisin protects macrophages from oxidized low density lipoprotein-induced apoptosis by inhibiting the endoplasmic reticulum stress pathway. Saudi Journal of Biological Sciences, 25(5), 849-857.

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Quantifying Gene Expression in Eucalyptus Leaves

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The total RNA was isolated from the young, mature, and senescent leaves of E. urophylla using an RNAprep Pure Plant Plus Kit (Tiangen, China, cat DP441). Approximately 1.5 μg of RNA was reverse-transcribed into first-stand cDNA using a cDNA synthesis kit (Tiangen, China, cat KR106). The qRT-PCR reaction mixture consisted of 10 μl of 2 × TransStart^® Tip Green qPCR SuperMix (TransGen Biotech, China, cat AQ101), 0.6 μl of forward primer, 0.6 μl of reverse primer, 2 μl of cDNA template, 0.4 μl of 50 × ROX reference dye, and 6.4 μl of RNase-free ddH₂O. qRT-PCR was performed on the Applied Biosystems 7500 Fast Real-Time PCR system for 39 cycles under the following conditions: 95°C for 15 min of pre-degeneration, 95°C for 10 s of degeneration, 58°C for 30 s of annealing, and 72°C for 30 s of extension. The samples were then heated to 95°C for 15 s and then 60°C for 1 min for dissolution curve analysis. Three technical replicates and three biological replicates were used for each sample and randomly selected gene. EuGADPH was a reference gene, and the primers used for qRT-PCR analysis are listed in Supplementary Table 1.

Liu Z., Wang J., Qiu B., Ma Z., Lu T., Kang X, & Yang J. (2022). Induction and Characterization of Tetraploid Through Zygotic Chromosome Doubling in Eucalyptus urophylla. Frontiers in Plant Science, 13, 870698.

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