Iscript dna synthesis kit

Manufactured by Bio-Rad

Sourced in United States, Germany

The IScript DNA synthesis kit is a versatile tool for the in vitro synthesis of DNA. It provides the necessary components and protocols to perform DNA synthesis reactions, enabling the generation of desired DNA sequences.

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IScript (787 citations) IScript kit (577 citations) IScript synthesis kit (80 citations) IScript complementary DNA synthesis kit (43 citations) IScript complementary DNA (cDNA) synthesis kit (21 citations) IScript genomic DNA Clear cDNA Synthesis Kit (2 citations)

32 protocols using iscript dna synthesis kit

Quantifying Myogenic Regulatory Factors in Cultured Cells

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Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to
analyze the expression level of actinin, myogenin, and MyoD. The cDNA was
directly synthesized from cultured cells using an iScript^TM DNA
synthesis kit (BioRad, US). Real-time RT-PCR using SensiMix^TMSYBR^® Hi-ROX Kit (Biokine, US) was performed with an AB 7500
Real-Time PCR System (Life Technologies, US). Thermocycling conditions were as
follows: 95°C for 5 min, 40 cycles of denaturation (15 s, 90°C), annealing (15
s, 55°C), and extension (15 s, 72°C). The primer sequences are listed in Table 1. The
housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to
normalize the data using the 2 − ΔΔCt method (n = 4).

Jo S.B., Erdenebileg U., Dashnyam K., Jin G.Z., Cha J.R., El-Fiqi A., Knowles J.C., Patel K.D., Lee H.H., Lee J.H, & Kim H.W. (2020). Nano-graphene oxide/polyurethane nanofibers: mechanically flexible and myogenic stimulating matrix for skeletal tissue engineering. Journal of Tissue Engineering, 11, 2041731419900424.

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Inflammatory Gene Expression of hPDLSCs

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Inflammatory gene expression of hPDLSCs was identified under extract media treatment. The hPDLSCs were plated in 6-well plates at 3.2 × 10⁴ cells/well conditions. After 24 h of stabilization, the cells were treated with various root canal sealer extracts for 72 h. After incubation, the total RNA of each sample was isolated with Ripospin^TM RNA isolation kit (GeneAll Biotechnology, Seoul, South Korea) and reverse transcription from extracted RNA was conducted using an iScript ^TM DNA synthesis kit (Bio-Rad, Hercules, CA, the United States). SensiMix^TM SYBR^® Hi-ROX Kit (Bioline, London, the United Kingdom) was utilized for Real-time PCR amplification with an AB 7500 Real-Time PCR System (Life Technologies, Carlsbad, CA, the United States). The PCR conditions were as follows: initial incubation at 95 °C for 5 min, followed by 40 cycles of 90 °C for 15 s, 55 °C for 15 s, and 72 °C for 15 s. The primer sequences are listed in Table 2. The relative gene expression levels were calculated using the 2 − ΔΔCt method (n = 4).

Jo S.B., Kim H.K., Lee H.N., Kim Y.J., Dev Patel K., Campbell Knowles J., Lee J.H, & Song M. (2020). Physical Properties and Biofunctionalities of Bioactive Root Canal Sealers In Vitro. Nanomaterials, 10(9), 1750.

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Quantifying autophagy and Wnt5a gene expression

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As previously described RNA was extracted using Trizol (Invitrogen) and the RNeasy Mini kit (Qiagen) (36 (link)). cDNA was then prepared using the iscript DNA synthesis kit (Bio-Rad, cat no. 1708891). SYBR Green dye-based PCR amplification was used to measure gene expression; qPCR was performed using the ABI StepOnePlus apparatus. The mRNA levels of samples were normalized with the mRNA levels of 18S using Universal 18S primers (Invitrogen, cat. No. AM1718). mRNA expression was determined using the standard curve method recommended by the manufacturer’s protocol (Perkin Elmer, Waltham, MA). Primers used are: ATG5 Forward, 5’ GGC CAT CAA TCG GAA ACT CAT 3’; ATG5 Reverse, 5’ AGC CAC AGG ACG AAA CAG CTT 3’; ATG12 Forward, 5’ TAG AGC GAA CAC GAA CCA TCC 3’; ATG12 Reverse, 5’ CAC TGC CAA AAC ACT CAT AGA GA 3’; WNT5A Forward, 5’ ATT CTT GGT GGT CGC TAG GTA 3’; WNT5A Reverse, 5’ CGC CTT CTC CGA TGT ACT GC 3’.

Ndoye A., Budina-Kolomets A., Kugel CH I.I.I., Webster M., Kaur A., Behera R., Rebecca V., Li L., Brafford P., Liu Q., Gopal Y.N., Davies M.A., Mills G.B., Xu X., Wu H., Herlyn M., Nicastri M., Winkler J., Soengas M.S., Amaravadi R., Murphy M, & Weeraratna A.T. (2017). ATG5 mediates a positive feedback loop between Wnt signaling and autophagy in melanoma. Cancer research, 77(21), 5873-5885.

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Extraction and Quantification of RNA

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Total RNA was prepared from cells using RNeasy Plus Mini Kit (QIAGEN) following manufacturer's protocol. RNA concentration was measured using NanoDrop (Wilmington, DE, USA). Reverse transcription was performed using iScript DNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and qRT-PCR reactions were performed using ABI Fast SYBR Green Master Mix (Life Technologies, Grand Island, NY, USA) as previously described [36 (link)].

Lee M., Beggs S.M., Gildea D., Bupp S., Lichtenberg J., Trivedi N.S., Hu Y., Bodine D.M, & Crawford N.P. (2015). Necdin is a breast cancer metastasis suppressor that regulates the transcription of c-Myc. Oncotarget, 6(31), 31557-31568.

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RNA Extraction from Small Intestine of Infected Mice

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Small intestine was excised from non-infected and H. diminuta-infected WT and IL-22^-/- mice, flushed with 4°C PBS, and the 3 cm portion of mid-intestine was cut in three pieces, placed in 1ml of TRizol Reagent (Invitrogen, California, USA) and homogenized for 60 seconds (Polytron MR2100, Kinematica AG, Switzerland). The RNA was extracted with chloroform/ethanol as previously [25 (link)] and 1 μg of RNA was used as the template for cDNA generation with the iScript DNA synthesis kit (Bio-Rad, USA). Conditions for the PCR were denaturation 95°C for 2 min, 40 amplifying cycles of 95°C 15 sec, 55°C 15 sec, 68°C 20 sec and final temperature 4°C; primer sequences are presented in S10 Fig.

Reyes J.L., Fernando M.R., Lopes F., Leung G., Mancini N.L., Matisz C.E., Wang A, & McKay D.M. (2016). IL-22 Restrains Tapeworm-Mediated Protection against Experimental Colitis via Regulation of IL-25 Expression. PLoS Pathogens, 12(4), e1005481.

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Quantification of Macrophage Inflammatory Markers

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RNA was extracted from macrophages lysate or infrarenal aorta lysate using RNeasy kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized with iScriptDNA synthesis kit (Bio-Rad). qPCR amplification was performed by using IQ SYBR Green Supermix (Bio-Rad) according to the manufacturer’s instructions. Each reaction was performed in duplicate. The result was expressed as the ratio of target gene versus GAPDH. Primers for qPCR were as follows: mTNF-α: 5′-TCT TCT CAT TCC TGC TTG TGG-3′(Forward) 5′-GGT CTG GGC CAT AGA ACT GA-3′(Reverse); mIL-1β: 5′-GAG TGT GGA TCC CAA GCA AT-3′(Forward) 5′-ACG GAT TCC ATG GTG AAG TC-3′(Reverse); mGAPDH: 5′-AGG TCG GTG TGA ACG GAT TTG-3′(Forward) 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′(Forward).

Zhang C., Hsu C.G., Mohan A., Shi H., Li D, & Yan C. (2020). Vinpocetine Protects Against the Development of Experimental Abdominal Aortic Aneurysms. Clinical science (London, England : 1979), 134(22), 2959-2976.

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Optimized qRT-PCR with Ascorbic Acid

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Cells were plated for 24 hours and then supplemented with 50 μg/mL L-ascorbic acid (Sigma-Aldrich) for a further 24 hours prior to lysis. RNA was isolated using Tri-Reagent (Sigma-Aldrich) using the manufacturer’s instructions, then reverse transcribed using the iScript DNA synthesis kit (Bio-Rad). qRT-PCR was run using primers at 900 nM (Table 1), and results were normalized to the endogenous control, 18S (Thermo Fisher Scientific, primer pair used at 100 nM). Primer sequences were obtained from the Harvard Primer Bank (https://pga.mgh.harvard.edu/primerbank/) and verified using Primer BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), or designed using Primer BLAST.

Jones C.E., Sharick J.T., Colbert S.E., Shukla V.C., Zent J.M., Ostrowski M.C., Ghadiali S.N., Sizemore S.T, & Leight J.L. (2021). Pten regulates collagen fibrillogenesis by fibroblasts through SPARC. PLoS ONE, 16(2), e0245653.

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Quantitative PCR Analysis of Macrophage Genes

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RNA was isolated using RNeasy Mini Kit (Qiagen) and cDNA was synthesized using iScript DNA Synthesis Kit (Bio-Rad). The cDNA was amplified by specific primers using FastStart Universal SYBR Green Master (Roche) and analyzed by the LightCycler 96 System (Roche). Expression levels in BMDMs and peritoneal macrophages were normalized to Hprt. The following primers were used for amplification: Cpt2 forward, 5′-CAGACAGTGGCTACCTATG AATCCT-3′, and reverse, 5′-TGGTCAGCTGGCCATGGTATTT GGA-3′; Arg1 forward, 5′-CTCCAAGCCAAAGTCCTTAGAG-3′, and reverse,5′-AGGAGCTGTCATTAGGGACATC-3′; Mgl2 forward, 5′-GCATGAAGGCAGCTGCTATTGGTT-3′, and reverse,5′- TAGGCCCATCCAGCTAAGCACATT-3′; Retnla forward, 5′-CCAATCCAGCTAACTATCCCTCC-3′, and reverse,5′-ACCCAGTAGCAGTCATCCCA-3′; Hprt forward, 5′-TTTCCCTGGTTAAGCAGTACAGCCC-3′, and reverse, 5′-TGGCCTGTATCCAACACTTCGAGA-3′.

Nomura M., Liu J., Rovira I.I., Gonzalez-Hurtado E., Lee J., Wolfgang M.J, & Finkel T. (2016). Fatty acid oxidation in macrophage polarization. Nature immunology, 17(3), 216-217.

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Androgen Receptor Expression Analysis

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RNA was extracted from control and ASR600-treated cells using the RNAeasy Mini Kit (Qiagen, Hilden, Germany). This was followed by reverse transcription using the iScript DNA Synthesis Kit (Bio-Rad, Hercules, CA). RT-PCR using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) with specific AR and PSA primers as per the protocol described previously (Dahiya et al., 2018 (link)). β-actin was used as the internal control.

Chandrasekaran B., Tyagi A., Saran U., Kolluru V., Baby B.V., Chirasani V.R., Dokholyan N.V., Lin J.M., Singh A., Sharma A.K., Ankem M.K, & Damodaran C. (2023). Urolithin A analog inhibits castration-resistant prostate cancer by targeting the androgen receptor and its variant, androgen receptor-variant 7. Frontiers in Pharmacology, 14, 1137783.

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Quantitative Gene Expression Analysis

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Tissue culture cell RNA were isolated using the RNAqueous kit whereas pancreatic and liver RNA were isolated using the ToTALLY RNA kit (Ambion, Carlsbad, CA). The Turbo DNA-free DNAse Treatment Kit (Ambion, Carlsbad, CA) was then used to remove trace genomic DNA followed by cDNA generation using the iScript DNA Synthesis Kit (Bio-Rad, Hercules, CA). Gene expression was then quantitated by PCR using the dUTP-containing FastStart SYBR Green Master Mix in conjunction with Uracil-DNA-Glycosylase (Roche, Nutley, NJ). Fold induction of gene expression was calculated using the 2(-ΔΔC(T)) method (Livak and Schmittgen 2001 (link)). PCR primer sequences are provided in Supplemental Material. The efficiency of primer amplification was tested in cDNA dilution analyses. These showed that correlation coefficients were ~0.99 and PCR efficiency was ~95%.

Syring K.E., Bosma K.J., Goleva S.B., Singh K., Oeser J.K., Lopez C.A., Skaar E.P., McGuinness O.P., Davis L.K., Powell D.R, & O’Brien R.M. (2020). Potential Positive and Negative Consequences of ZnT8 Inhibition. The Journal of endocrinology, 246(2), 189-205.

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