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

Reduction of target expression was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the StepOnePlus System (Thermo Fisher). Briefly, RNA was extracted from ∼50–100 mg of liver tissue from each mouse using a 96-well format RNeasy Kit (Qiagen) or the PureLink Pro 96 total RNA Purification Kit (Thermo Fisher) with on column DNase I (Thermo Fisher) treatment and target mRNA was measured by RT-qPCR using the Express StepOne SuperMix Kit (Thermo Fisher). Part of the initial total RNA eluates were diluted to a concentration of approximately 10 ng/μl and the actual concentration was determined using the Quant-IT Ribogreen RNA assay (Thermo Fisher). Approximately 50 ng of total RNA was combined with 10 μl Express PCR Supermix, target specific forward and reverse primers and hydrolysis probe (at 375, 375 and 125 nM, respectively; Integrated DNA Technologies), 0.4 μl ROX Reference Dye and 2 μl of Express Superscript Mix in a total reaction volume of 20 μl in a 96-well PCR plate. Samples were run on StepOnePlus thermocyclers with the following cycling protocol: 15 min at 50°C, 2 min at 95°C and 40 cycles of 15 s at 95°C and 1 min at 60°C. C values were derived using the StepOne software v2.3 and relative levels of mRNA expression for each sample was determined by comparing the sample C to the C values of a dilution series (serial 2-fold or 4-fold dilutions) of concentrated RNA, from a pool of the initial RNA eluates. Relative target expression was then normalized to total input RNA levels based on the Ribogreen assay values. The normalized sample values were then divided by the average value of all samples from the appropriate control group to generate the reported relative expression of the target mRNA. Data are mean values ± standard deviations.

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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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