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

RNAs were isolated using TRIzol (Thermo Fisher Scientific) or RNeasy 96 Kits (Qiagen) according to protocols supplied by the manufacturers. TaqMan One-step qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents (Thermo Fisher Scientific). Briefly, total RNAs (equation M150–100 ng) in 5 μl nuclease-free water were mixed with 0.3 μl primer probe sets containing forward and reverse primers (10 μM of each) and fluorescently labeled probe (3 μM), 10 μl 2 × RT-PCR Buffer, 0.8 μl 25 × RT-PCR Enzyme Mix, 1.7 μl Detection Enhancer and 2.2 μl nuclease-free water in a 20 μl reaction. Reverse transcription was performed at 45°C for 10 min, the reactions were then denatured at 95°C for 10 min, and forty cycles of PCR reactions were conducted at 95°C for 15 s and 60°C for 60 s within each cycle, using Applied Biosystems StepOnePlus Real-Time PCR system. Expression levels of target RNA were normalized to total RNA quantified using Quant-iT RiboGreen RNA Reagent (Thermo Fisher Scientific). Primer-probe sets for human Rnaseh1 (Hs00161407_m1), human TOP1 (Hs00243257_m1), mouse Cdkn1a (Mm01303209_m1), and mouse Tnfrsf10b (Mm00457866_m1) mRNAs were purchased from Thermo Fisher Scientific. Primer sequences for quantification of NCL1 mRNA precursor (NCL1 pre-mRNA) are: forward, 5′-CTC TGT CAC TGG TAT CTT TTC CC-3′; reverse, 5′-CAA AAC CAA ACC TAG AAC ACC AAA TG-3′; and probe, 5′-CAA GGC TAC TTT CTG TGG GAT GGC T-3′. Expression levels of 47S pre-rRNA precursor and mature 18S rRNA were examined using EXPRESS One-Step SYBR GreenER Kit (Thermo Fisher Scientific). Levels of 47S pre-rRNA precursor were normalized to the levels of 18S rRNA. Primer sequences for 47S pre-rRNA are: forward, 5′-TGT CAG GCG TTC TCG TCT C-3′ and reverse, 5′-AGC ACG ACG TCA CCA CAT C-3′. Primer sequences for 18S rRNA are: forward, 5′-GCA ATT ATT CCC CAT GAA CG-3′ and reverse, 5′-GGG ACT TAA TCA ACG CAA GC-3′. Northern analysis was performed as described previously (38). Briefly, total RNAs (5 μg) were resolved on a 1% agarose gel buffered with 1 × MOPS and transferred onto a nylon membrane. Hybridization was performed at 42°C using 5′-end [γ-32P] labeled oligonucleotide probes. The probe for detecting pre-rRNA spans the boundary between 18S and ITS1. Probe sequence: 5′-CCT CGC CCT CCG GGC TCC GGG CTC CGT TAA TGA TC-3′.

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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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