Qubit RNA HS Assay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
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

DNA contaminations from extracted total RNA were removed with the use of DNAse I (Fermentas) treatment following the manufacturer׳s recommendations. RNA concentration was determined by fluorometer Qubit 2.0 using Qubit RNA HS Assay Kit (Thermo Fisher). Transcriptomic libraries were constructed using Ion Total RNA-Seq Kit v2 (Life Technologies) with the following modifications of the protocol. For RNA fragmentation 1 µl of 10x RNase III buffer (Life Technologies) was added to 9 µl of RNA solution and heated for 10 min at 95 °С followed by immediate snap-cooling on ice. After that 1 µl of 10 µM ATP and 1 µl of polynucleotide kinase (Fermentas) were added to the solution from the previous step and the whole mix was incubated at 37 °С for 30 min. Fragmented RNA was cleaned up using Micro Bio-Spin Chromatography Columns (Bio-Rad). Further steps of library preparation including adapter ligation, first-strand cDNA synthesis, and amplification were carried out in accordance with manufacturer׳s instructions. Fragments of cDNA corresponding to rRNA were depleted using duplex-specific nuclease (Evrogen) as described in [2] with the following modifications. Only one round of DSN-normalisation was performed and the re-annealing of ds-cDNA at 68 °C was carried out overnight. The prepared library was purified by magnetic beads Agencourt AMPure XP (Beckman Coulter Inc.) and its quality was assessed by 2100 Bioanalyzer (Agilent Genomics) using Agilent High Sensitivity DNA Kit (Agilent Genomics).

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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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