Quant-iT™ RNA Assay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Quantitation Range is 5-100 ng	Prepare the Quant-iT™ RNA working solution by diluting the Quant-iT™ RNA reagent 1:200 in Quant-iT™ buffer. Incubate the tubes for 2 minutes at room temperature.

Upstream tips
Quantitation Range is 5-100 ng
Protocol tips
Prepare the Quant-iT™ RNA working solution by diluting the Quant-iT™ RNA reagent 1:200 in Quant-iT™ buffer. Incubate the tubes for 2 minutes at room temperature.

Publication protocol

The Ovation RNA-Seq method was employed for cDNA synthesis according to the manufacturer’s instructions (NuGen, San Carlos, CA). cDNA was then concentrated and suspended in 10 mM Tris-HCl (pH 8) using MinElute spin columns (Qiagen, Valencia, CA). cDNA was quantified using the Quant-iT dsDNA HS Assay (Life Technologies Corporation), whereas the molecular weight distribution was determined using the BioAnalyzer 2100 and the Agilent DNA 7500 Chip Assay (Agilent Technologies, Santa Clara, CA). A total of 500 ng of cDNA (10ng/μl) was used for Illumina library construction with the Illumina paired-end LT indexing protocol as previously published [38], [39]. Briefly, 500 ng of each library was hybridized with the Agilent mouse exome reagent [40]. Eight mouse sample sets were sequenced from independently generated biological replicates that included four samples of FM microglia and four samples of tumor-associated microglia. Corresponding RNA-Seq paired-end reads were processed using the TopHat suite [41] with Cufflinks [42], [43], [44]. All RNA-sequencing data will be uploaded into Gene Expression Omnibus (GEO) database.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Quant-iT™ RNA Assay Kit below.

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