Quant-iT™ RiboGreen™ RNA Assay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
This kit enables quantitation of as little as 1 ng/mL.	Add 1.0 mL of the aqueous working solution of the Quant-iT™ Ribo Green® reagent and Incubate for 2 to 5 minutes at room temperature, protected from light.

Upstream tips
This kit enables quantitation of as little as 1 ng/mL.
Protocol tips
Add 1.0 mL of the aqueous working solution of the Quant-iT™ Ribo Green® reagent and Incubate for 2 to 5 minutes at room temperature, protected from light.

Publication protocol

Total RNA was extracted from cell lines using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) and from tissues using RNA-Solv Reagent (Omega Bio-Tek, Norcross, GA) according to the manufacturers’ instructions. RNA concentration was qualified and quantified by UV absorption spectrophotometry and the Quant-iT RiboGreen RNA Assay Kit (Life Technologies, Carlsbad, CA). Reverse-transcription of total RNA was performed using Moloney murine leukemia virus reverse-transcriptase with oligo-dT and random hexamers as previously described (Hoshimoto et al., 2012; Koyanagi et al., 2010). The qPCR of CD44 standard, CD44v6, ESRP1, ESRP2, PTBP1, and U2AF2 mRNAs was performed on the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) by using the primers described in the Supplemental Table S2. The amount of cDNA was normalized using SDHA mRNA (Supplemental Table S2) expression as an internal control. A melanocyte cell line (HEMn-LP, Life Technologies, Carlsbad, CA) was used as an external reference for gene expression. RNA extracted from the established melanoma cell line LF-0023 was used as an internal control to normalize the variation in qPCR assays. qPCR data was analyzed using the ddCq method as previously described (Heid et al., 1996).

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Quant-iT™ RiboGreen™ RNA Assay Kit below.

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