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

Frozen tissues, weighing 100–200 mg, were placed in Lysing Matrix D Fast-Prep Tubes (MP Biomedicals) containing 500 μl ice cold detergent-free buffer (10 mM Tris pH 7.4, 100 mM NaCl, 2.5 mM MgCl) supplemented with 100 μg/ml cycloheximide (EMD) and EDTA-free HALT® protease inhibitor cocktail (ThermoFisher). Samples were homogenized in a tissue homogenizer, shaking at 2000 oscillations/min for 60–120 s. Resulting homogenates were cleared by centrifugation at 1000xg for 10 min at 4°C. Supernatants were then centrifuged twice at 16 000xg for 10 min at 4°C. Resulting S16 lysates were layered on top of 5–60% sucrose gradients and spun in an XL-90 ultracentrifuge at 40 000 rpm for 1.5 h using a SW41 rotor (Beckman Coulter). Gradients were fractionated into 15 or 8 equal volumes by either a piston gradient fractionater (BioComp Instruments) or STAR-series liquid handling robot (Hamilton). Polysome-containing fractions were confirmed by in-line UV (254 nm) measurements or Quant-iT® RiboGreen RNA reagent (Life Technologies). Polysome-containing fractions were then analyzed using miRNA TaqMan assays (Life Technologies) to quantify relative levels of the miR-of-interest (MOI; i.e. the miRNA being inhibited) and a reference (REF) miRNA. Relative displacement of the MOI normalized to the REF was then calculated between treated and control samples using the ΔΔCT method. Displacement values are reported in log scale such that positive values reflect loss of miRNA from polysome fractions (i.e. displacement = ΔΔCT)

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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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