Influence of low-input RNA from FF and FFPE samples on gene expression quantification by NanoString, microarray and RNA-seq: a comparative study [microarray]

Purpose: The goals of this study is to determine the best method of gene expression quantification (RNA-seq, Microarray, NanoString) and amplification kits adapted to low-input and/or low-quality RNA samples (FFPE samples) Methods: Mouse bladder cancer cell line (mouse bladder cancer cell line, BC57) and mouse normal mouse normal urothelium were fixed in formalin and embedded in paraffin (FFPE), andfesh frozen (FF) in liquid nitrogen. The total RNA of these 4 samples were tested in triplicate by 3 technologies (NanoString, RNA-seq and Microarray) and the results were compared to its reference (high-quality and high-input RNA of mouse bladder cancer cell line and mouse normal mouse normal urothelium). For microarray technology, each sample was tested by two amplification kits: GeneChip Pico WT and SensationPlus with 2 input quantities: 500pg and 2ng of total RNA, except for the SensationPlus which the recommended quantity is 50ng of total RNA. Microarray data, obtained with the kit GeneChip WT Plus from 100ng of total RNA from fresh frozen samples, was considered as the reference. All samples were hybridized on MTA 1.0 microarrays. Results obtained with the two tested kits were compared to those from GeneChip WT Plus. To determine which is the best kit suitable for microarray from low-input and low-quality RNA samples, we performed microarray control quality metrics, principal component analysis, and a differential analysis between the mouse bladder cancer cell lines and the mouse normal mouse normal urothelium for each input quantity, amplification kit and method of sample preservation (FF or FFPE). Results: According to our results, the GeneChip Pico kit are recommended for quantification of gene expression of FFPE and FF samples from 2ng of total RNA. This kit is not recommended for samples below 2ng of total RNA. The kit SensationPlus is recommended for FFPE samples at 50ng of total RNA. For microarray technology, 4 samples were tested by two amplification kits: GeneChip Pico WT and SensationPlus with 2 input quantities: 500pg and 2ng of total RNA, except for the SensationPlus which the recommended quantity was 50ng of total RNA. Microarray data, obtained with the kit GeneChip WT Plus from 100ng of total RNA from fresh frozen samples, was considered as the reference. Each Sample tested with the GeneChip PicoWT kit was done in triplicate and each sample tested with SensationPlus and GeneChip WT Plus was done in duplicate.