Iscript kit

Manufactured by New England Biolabs

The iScript kit is a reagent system for reverse transcription of RNA to cDNA. It contains the necessary components for efficient conversion of RNA to first-strand cDNA suitable for use in various downstream applications such as real-time PCR, endpoint PCR, and gene expression analysis.

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Lab products found in correlation

Trizol, by Thermo Fisher Scientific (2 mentions) Dnase 1, by New England Biolabs (2 mentions) Cfx connect qpcr detection system, by Bio-Rad (2 mentions) Luna universal qpcr master mix, by New England Biolabs (2 mentions)

Spelling variants of this product

IScript cDNA Synthesis kit (3 citations)

2 protocols using iscript kit

SARS-CoV-2 Genomic RNA Quantification

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Genomic RNA was extracted from cell pellets using Trizol (Ambion), according to manufacturer instructions. RNA was treated with DNAse I (NEB) and used as a template to reverse-transcribe cDNA by Iscript kit (NEB). qPCR reactions were done using the Luna Universal qPCR Master Mix (NEB) and a CFX connect qPCR Detection System (BioRad). To determine the number of vRNA copies per mL, plasmids containing the nucleocapsid gene of SARS-CoV-2 (cloned from the USAWA1/2020 isolate) were used as standards and diluted serially 10-fold to determine target copy numbers. Threshold cycle (Ct) values were plotted against the number of target copies, and the resultant standard curve was used to determine the number of genome equivalents of vRNA in the samples. For cell pellet samples, the vRNA copy number was normalized to the housekeeping gene HRT1. All samples were within the range of linearity of a standard curve, and the primer efficiencies were 100% +/− 5%. The primer sequences targeting nucleocapsid were: 5′- TCCTGGTGATTCTTCTTCAGG-3′ and 5′-TCTGAGAGAGGGTCAAGTGC-3′. HRT1 primers sequences are: 5′-GGTCCTTTTCACCAGCAAGCT-3′ and 5′-TGACACTGGCAAAACAATGCA-3′.

Wu C.T., Lidsky P.V., Xiao Y., Cheng R., Lee I.T., Nakayama T., Jiang S., He W., Demeter J., Knight M.G., Turn R.E., Rojas-Hernandez L.S., Ye C., Chiem K., Shon J., Martinez-Sobrido L., Bertozzi C.R., Nolan G.P., Nayak J.V., Milla C., Andino R, & Jackson P.K. (2022). SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming. Cell, 186(1), 112-130.e20.

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SARS-CoV-2 Viral RNA Quantification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Genomic RNA was extracted from cell pellets using Trizol (Ambion), according to manufacturer instructions. RNA was treated with DNAse I (NEB) and used as template to reverse-transcribe cDNA by Iscript kit (NEB). qPCR was done using the Luna Universal qPCR Master Mix (NEB) and a CFX connect qPCR Detection System (BioRad). To determine the number of vRNA copies per mL, plasmids containing the nucleocapsid gene of SARS-CoV-2 (cloned from the USA-WA1/2020 isolate) were used as standards and diluted serially 10-fold to determine target copy numbers. Threshold cycle (Ct) values were plotted against the number of target-copies, and the resultant standard curve was used to determine the number of genome equivalents of vRNA in the samples. For cell pellet samples, the vRNA copy number was normalized to the housekeeping gene HRT1. All samples were within the range of linearity of a standard curve, and the primer efficiencies were 100% +/− 5%. The primer sequences targeting nucleocapsid were: 5′-TCCTGGTGATTCTTCTTCAGG-3′ and 5′-TCTGAGAGAGGGTCAAGTGC-3′. HRT1 primers sequences are: 5′-GGTCCTTTTCACCAGCAAGCT-3′ and 5′-TGACACTGGCAAAACAATGCA-3′.

Yang J., Xiao Y., Lidsky P.V., Wu C.T., Bonser L.R., Peng S., Garcia-Knight M.A., Tassetto M., Chung C.I., Li X., Nakayama T., Lee I.T., Nayak J.V., Ghias K., Hargett K.L., Shoichet B.K., Erle D.J., Jackson P.K., Andino R, & Shu X. (2023). Fluorogenic reporter enables identification of compounds that inhibit SARS-CoV-2. Nature microbiology, 8(1), 121-134.

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