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 sequencing library construction, RNA was extracted from frozen cell suspensions (Patients 1, 2, 3, 7, 9, and 10) or FFPE samples (Patients 4, 5, 6, 8 and 10) using Qiagen RNeasy Mini kit or Qiagen FFPE RNeasy kit, respectively. RNA-Seq libraries were prepared using Illumina TruSeq Stranded mRNA Library Prep Kit (for cell suspensions) or Illumina’s TruSeq RNA Access Library Prep Kit (for FFPE samples). Total RNA was quantified using the Quant-iT™ RiboGreen® RNA Assay Kit and normalized to 5ng/µl. For Patients 1, 2, 3, 7 and 9, each sample was transferred into library preparation which was an automated variant of the Illumina TruSeq™ Stranded mRNA Sample Preparation Kit. This method preserves strand orientation of the RNA transcript. It uses oligo dT beads to select mRNA from the total RNA sample. It is followed by heat fragmentation and cDNA synthesis from the RNA template. The resultant 500bp cDNA then goes through library preparation (end repair, base ‘A’ addition, adapter ligation, and enrichment) using Broad designed indexed adapters substituted in for multiplexing. For Patient 4, the mRNA enrichment step was omitted prior to library preparation. The resulting libraries were quantified with qPCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms and then pooled equimolarly. For Illumina Sequencing, pooled libraries were normalized to 2nM and denatured using 0.1 N NaOH prior to sequencing. Flowcell cluster amplification and sequencing were performed according to the manufacturer’s protocols using either the HiSeq 2000 or HiSeq 2500. Each run was a 101bp paired-end with an eight-base index barcode read. Data was analyzed using the Broad Picard Pipeline which includes de-multiplexing and data aggregation.

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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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