Quant-iT™ RiboGreen™ RNA Assay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Treat samples with Proteinase K before RNA extraction. 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
Treat samples with Proteinase K before RNA extraction. 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

RNA was extracted from 1 mL of plasma using the miRCURY RNA Isolation Kit for Biofluids (Exiqon) according to manufacturer’s protocol except for noted modifications. 1 μL of 20 mg/mL glycogen (Roche) was added to plasma prior to RNA extraction. All samples were treated with T4 polynucleotide kinase (New England Biolabs) to facilitate 5’ hydroxyl terminus phosphate labelling and allow greater binding of adaptors during library preparation, and RNA sample volume was reduced to ≤10 μL in a standard lyophilizer at room temperature. RNA concentration was determined by Quant-iT RiboGreen RNA Assay Kit.

Prior to RNA extraction, a subset of plasma samples were treated with Proteinase K (PK; 100 μg/mL in 0.5% SDS solution) for 30 min at 50°C. Samples were treated in one of three groups: 1) no PK treatment; 2) PK treatment prior to addition of guanidinium thiocyanate (GITC) containing lysis buffer (from miRCURY RNA Isolation Kit); 3) PK treatment following the addition of GITC-containing lysis buffer. Where indicated, ribodepletion was performed using the Ribo-Zero Magnetic Gold kit (Epicentre) according to manufacturer’s protocol. A schematic summarizing plasma sample treatments is shown in Fig 1

For samples that were analyzed by digital droplet PCR (ddPCR), ethanol precipitation was performed to remove any excess SDS solution that could inhibit the PCR reaction. Briefly, 0.3 M NaCl, and 3x volumes of 100% ethanol were added to the solution and the RNA precipitated at -80°C overnight. Samples were then pelleted by centrifugation, washed with 80% ethanol, and re-suspended in 10 μL RNAse-free water.

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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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