Total RNA Prep
- RNA must be extracted from specimens using appropriate laboratory procedures to result in high quality RNA suitable for subsequent analyses. In our experience, outstanding results can be obtained with the Qiagen RNeasy and family of kits. We have also had success processing RNA isolated using TRIzol reagent when followed by further purification using the RNeasy Kit. We do not recommend RNA purification using TRIzol reagent alone due to the potential for contamination with organic solvents and proteins, which may often interfere with downstream assays. The use of co-precipitants such as tRNA, glycogen, Pellet Paint® or linear acrylamide should be avoided. Some assays such as Small RNA Sequencing require Total RNA that has been isolated using procedures that do not exclude low molecular weight RNAs, such as the Qiagen miRNeasy kit.
- Highly purified and intact total RNA in pure, nuclease-free water will always produce the best results and is required for most applications. RNA concentration should be determined using spectrophotometry or fluorescent stains such as RiboGreen®. The A260/A280 ratio of the RNA specimen should approach 2.0 and exceed 1.6. The integrity of the specimen should be assessed using an Agilent 2100 Bioanalyzer (RIN ≥ 7.0) or a Caliper LabChip® GX (RQS ≥ 6.4). Total RNA samples that are isolated at Expression Analysis or received directly will be evaluated using these same criteria, and you will be notified when specimens are non-conforming.
- When submitting RNA specimens for target preparation and analysis, the amount and concentration of RNA required will vary according to the selected laboratory procedure. Our specimen requirements are designed to ensure that ample material is available for quantitation and quality assessment of the incoming RNA, with concentration values matched to the requirements of the assay. Adherence ensures that delays resulting from dilution or concentration of the sample are minimized. Expression Analysis recognizes that these requirements may not be met in all situations. Your Service Consultant can work with you to accommodate special circumstances.
- For standard processing using Affymetrix 3’ Expression GeneChips, specimens requirements are 2.5 µg of total RNA at a concentration of 125 ng/µL in 20 µL total volume of pure, nuclease-free water. For standard processing using the Illumina Direct Hybridization Assay, specimen requirements are 250 ng of total RNA at a concentration of 25 ng/µL in 10 µL total volume of pure, nuclease-free water. Your Service Consultant can provide you with detailed requirements for alternative assays.