Optimization of RNA extraction from FFPE tissues for expression profiling in the DASL assay

Abstract

Formalin-fixed paraffin-embedded (FFPE) breast tumor tissues are readily available and represent a largely untapped, vast resource for molecular profiling of clinical samples with long-term follow-up data. We have optimized the conditions and parameters that result in the preparation of total RNA that is of the necessary quality for use in the DASL (cDNA-mediated annealing, selection, extension, and ligation) assay in which expression of 502 genes are analyzed simultaneously using as little as 100 ng of input RNA.

Figure 1. Quality control analysis of total RNA samples prepared using four commercially available kits

(A) Concentration of samples varied from sample to sample, and from kit to kit, but it was clearly tissue-dependent. The overnight incubation with Proteinase K increased the yield of total RNA across the methods tested. (B) The A260/A280 ratio was close to the ideal value of two, with the exception of the SuperArray kit. (C) The lowest Ct values were obtained using the Ambion and Roche kits, while the SuperArray kit provided the least amount of usable RNA. (D) Replicate reproducibility was highest in samples that were incubated overnight, with the best results obtained using the Ambion and Roche kits. (E) Representative Agilent 2100 Bioanalysis of the same patient (sample 4) using the Roche kit with overnight and 3hr Proteinase K digestion is shown. Median RNA size is approximately 100-200 nt.

Figure 2. Longer Proteinase K digestion increases the replicate reproducibility

The R² value between two replicates of the same sample was superior for samples that were treated with Proteinase K overnight than for the samples that were incubated with Proteinase K for only three hours or less. In addition, the replicate reproducibility of the samples incubated in Proteinase K for a shorter time was the best for the Ambion kit, then Qiagen, Roche, and finally SuperArray kit.

Figure 3. Samples cluster by patient regardless of the RNA isolation method used

(A) Clustering of all of the patients using all of the RNA samples, 8 per patient. In an initial analysis of data for the 6 patients obtained in the DASL assay, clustering of the samples grouped by patient regardless of the kit or method used. This suggests that the DASL assay can tolerate, to some extent, differences in RNA quality. (B) Scatterplots of gene expression patterns of samples from Patient 1 prepared with Ambion (A1) or Roche (R1) kits and of Patient 2 prepared with Ambion (A2) or Roche (R2) kits. The replicate reproducibility of these scatterplots indicates that there were significant differences in gene expression between patients 1 and 2 and high correlation of expression patterns for these patients' RNA prepared by the two different RNA extraction methods. (C) Hierarchical clustering of Z-score normalized data from six patients. Each column is the average of four DASL assays, two technical replicates prepared with the Ambion kit, and two technical replicates prepared with the Roche Kit. All four were prepared with an overnight Proteinase K digestion.