Gene expression assay 

For purification of total RNA and FFPE RNA, standard commercially available kits (Qiagen™/Ambion™) are recommended. Samples should be resuspended at a concentration between 20-30ng/μL in purification kit buffers or RNAse free water. The recommended volume of sample is 5μL. To adjust for pipetting errors, please supply minimum 6μL of sample. For samples with low concentrations, the NanoString protocol can be adapted to use 8μL of sample.

Cell lysates should be prepared in a GITC lysis buffer such as Qiagen™ buffer RLT at a concentration as below:

miRNA assay 

NanoString miRNA assays require purified total RNA as input material. NanoString recommends using 100ng of total RNA, extracted from any cell or tissue type including from FFPE material. NanoString recommends preparing samples with a concentration of >33 ng/μL. 3μL of sample are used in the assay. Please supply 4-5μL of sample to adjust for pipetting errors.

Unpurified lysates cannot be used for the assay. RNA quality is critical for the NanoString miRNA assay as lysis and RNA extraction contaminants can impact the assay performance by inhibiting the enzymatic ligation and purification steps. Typical lysis or extraction contaminants include guanidinium isothiocyanate, phenol, guanidinium HCl, and ethanol. 

Purified RNA quality can be gauged via a spectrophotometer. NanoString recommends an A260/A280 ratio of 1.9 or greater and an A260/A230 ratio of 1.8 or greater for optimal results.

CNV assay 

The nCounter CNV assays require purified, double-stranded genomic DNA. Accurate quantification is vitally important. DNA purification methods that leave a significant amount of residual RNA are to be avoided, as they may lead to over-estimation of DNA concentration when measured by UV absorbance. For DNA preparations, NanoString suggest an A260/A280 ratio of 1.8 and an A260/A230 ratio between 2.0 and 2.2 for optimal results.