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 the isolation and purification of messenger RNA (mRNA) from whole blood, the PAXgene extraction kit (Qiagen, Valencia, CA, USA) was used. The PAXgene tubes were stored at −20 °C and RNA was isolated within 6 months after sample collection according to the manufacturer's protocol, including an optional DNase digestion step. The total mRNA was quantified using a ribogreen assay (Invitrogen Quant-it Ribogreen Thermo Fisher Scientific, Walham, MA, USA). The quality of total RNA was determined using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). A threshold of RNA integrity number of 7 was applied to ensure good RNA quality. Genome-wide RNA expression profiling was obtained with the Illumina HumanRef-12 (version 3) arrays using Illumina's standard protocol at the UCLA Neuroscience Genomics Core. Raw data extraction and background correction were performed using GenomeStudio (Illumina, San Diego, CA, USA). Quality control was performed by assessment of hierarchical clustering, box plots, density distribution plots and pair-wise correlations (Supplementary Methods). The Lumi package in R was used for robust spline normalization and variance stabilizing transformation of the gene expression data.17, 18 A total of 48 803 probes were included on the Illumina microarray. To select those probes that were expressed in at least one sample, a gene filter was applied with the detection P-value generated set to 0.01 by GenomeStudio Software (Illumina).19 Our gene expression data set consisted of three batches based on Illumina serial numbers. Normalization between batches was performed with the ComBat package for R software.20

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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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