Qubit RNA HS Assay Kit

Protocol tips

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The Qubit® assays are designed to be performed at room temperature, as temperature fluctuations can influence the accuracy of the assay.	Dilute Qubit® RNA HS Reagent 1:200 in Qubit® RNA HS Buffer.

Upstream tips
The Qubit® assays are designed to be performed at room temperature, as temperature fluctuations can influence the accuracy of the assay.
Protocol tips
Dilute Qubit® RNA HS Reagent 1:200 in Qubit® RNA HS Buffer.

Publication protocol

Validation of the Spike-in Qubit™ RNA HS assay
The Qubit™ RNA HS Assay Kit (Life Technologies, Thermo Fisher Scientific Inc.), Qubit™ 2.0 Fluorometer (Life Technologies, Thermo Fisher Scientific Inc.) and Axygen PCR-05-C tubes (Axygen) were used for all measurements. The Qubit™ working solution was made according to manufacturer’s instructions. We added 180 μL of working solution to each assay tube, up to 20 μL of RNA, and water to bring the final volume to 200 μL. 10 μL of the Qubit™ RNA Standard solutions were used for standard tubes. “RNA spike-in” was made by diluting the Qubit™ RNA Standard #2 (10 ng/μL rRNA) included in the Qubit™ RNA HS Assay kit to 2.5 ng/μL and 2 μL was added into each tube. 18 μL of water was added into one tube for RNA spike-in alone reading. “RNA sample” was made by diluting the Qubit™ RNA Standard #2 to 250 pg/μL and increasing volumes of RNA sample were added into remaining tubes to create expected reading increases of 1, 2, 3, 4, 5, 7.5, 15 and 20 pg/μL over RNA spike-in alone. Assay tubes were vortexed for 2–3 s, centrifuged briefly (~5 s), and then incubated at room temperature for 2 min to allow the assay to reach optimal fluorescence before measuring with the Qubit™ Fluorometer. Each tube was measured three times to obtain the average reading. Reading increase was calculated by subtracting RNA spike-in reading from that of spike-in plus sample RNA reading. The experiment was repeated four times using independently prepared RNA spike-in, RNA sample, and working solution. In addition, the total RNA from human trophoblast cells was used as “RNA sample” and tested as described above for a total of four independent experiments.

Comparison between the Spike-in Qubit™ RNA HS Assay and Quant-iT™ RiboGreen® RNA Assay
Based on the original concentration measured by NanoDrop® ND-1000 (Thermo Fisher Scientific Inc.), the trophoblast total RNA was diluted to 60 pg/μL in water. A mixture of RNA and DNA (60 pg/μL each) was prepared by diluting in water the trophoblast total RNA and the Qubit™ DNA Standard #2 (10 ng/μL DNA) included in the Qubit™ DNA HS Assay kit. 18 μL of each sample was measured by the Spike-in Qubit™ as described above and by the Quant-iT™ RiboGreen® RNA Assay (Life Technologies, Thermo Fisher Scientific Inc.) following manufacturer’s instructions using a BioTek Synergy™ 4 Multi-Mode Microplate Reader (BioTek). Four independent repeats were performed with each method.

Quantification of plasma RNA samples
The standard Qubit™ RNA HS Assay was performed according to manufacturer’s instructions. The Spike-in Qubit™ RNA HS Assay was performed as described above. For the Spike-in Qubit™ Assay, RNA sample concentration was calculated as: [Sample] = reading increase (pg/μL) x assay volume (μL) ÷ sample volume for Spike-in Qubit™ (μL). Three independent measurements using separately prepared RNA spike-in and Qubit™ reagents were made for each sample. A detailed step-by-step protocol for the Spike-in Qubit™ RNA HS Assay is provided in the Supplementary Methods.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Qubit RNA HS Assay Kit below.

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