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

For RNA preparation, 5 μl of SUPERase In RNase Inhibitor (Ambion) was added to the supernatants per 10 ml media. The media was then filtered sequentially through the 2 μm filter (GE Healthcare, UK), 0.8 μm filter (EMD Millipore, MA), and 0.22 μm filter (EMD Millipore), with no/minimal pressure applied. The filtrate was split to 15 ml per sample and further filtered through the 0.02 μm filter (GE Healthcare) with up to 75 psi pressure applied. To facilitate the 0.02 μm filtration, a mechanical syringe pump was designed and manufactured (Supplementary Fig. 2). Upon filtration, each filter was washed with 1 ml DPBS (Corning), and the corresponding fractions were lysed with 600 μl lysis solution of the miRCURY RNA Isolation Kit—Cell & Plant (Exiqon, Denmark). The fractions collected on 0.02 μm filters were lysed with 900 μl lysis solution. The last flow-through fractions of 0.02 μm filters were pooled together (up to 30 ml) and concentrated ~60 times using 3 kDa Amicon Ultra Centrifugal Filters (EMD Millipore) at 4000×g, 4 °C, for 60 min. The concentrates were collected and lysed with six volumes of the same lysis solution (Exiqon). Total RNA was then isolated from all fractions as recommended by miRCURY protocol, with on-column DNase treatment (Qiagen, Germany). The corresponding 1.2 ml of the source neurospheres were span down at 300×g, 4 °C for 5 min, and total cellular RNA was isolated from them and analyzed in parallel. The same protocol was carried out for RNA isolation from fresh media, with 500 ml media input. For RNA isolation from primary cells cultured in 24-well plates, the cells were lysed with 350 μl lysis solution per well. The concentrations of cellular and extracellular RNA were determined using the NanoDrop 2000 Spectrophotometer and Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific), respectively. The RNA quality was examined using Agilent 2100 Bioanalyzer (Agilent, CA) and the RNA Integrity Number (RIN) estimated.

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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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