Prognostic gene expression signatures can be measured in tissues collected in RNAlater preservative

Abstract

Gene expression signatures have the ability to serve in both prognostic and predictive capacities in patient management. The use of RNA as the starting material and the lability of this analyte, however, dictate that tissues must be snap-frozen or stored in a solution that can maintain the integrity of the RNA. We compared pairs of snap-frozen and RNAlater preservative-suspended tissue from 30 such paired lymph node-negative breast tumors and 21 such paired Dukes' B colon tumors. We assessed the correlation of gene expression profiles and prediction of recurrence based on two prognostic algorithms. Tissues stored in RNAlater preservative generated expression profiles with excellent correlation (average Pearson correlation coefficients of 0.97 and 0.94 for the breast and colon tumor pairs, respectively) compared to those produced by tissues that were snap-frozen. The correlation in the prediction of recurrence was 97% and 95% for the breast and colon tumor pairs, respectively, between these two types of tissue handling protocols. This novel finding demonstrates that prognostic signatures can be obtained from RNAlater preservative-suspended tissues, an important step in bringing gene expression signatures to the clinic.

Figure 1

Box plot depicting the distribution in the Pearson correlation coefficients obtained from the 6 colon tumor samples performed in triplicate (18 pairwise comparisons), 34 pairwise comparisons for the lymph node-negative breast tumors (including repeats), and 25 pairwise comparisons of Dukes’ B colon tumors (including repeats). The line in the box indicates the median whereas the edges of the box indicate the 25th and 75th percentiles of the data.

Figure 2

Box plot depicting the distribution in the M values obtained from the 18 pairwise comparisons of the colon tumor replicates (A), 34 pairwise comparisons for the RNAlater preservative-suspended and snap-frozen breast tumors (B), and 25 pairwise comparisons of RNAlater preservative-suspended and snap-frozen colon tumors (C). The line in the box indicates the median whereas the edges of the box indicate the 25th and 75th percentiles of the data. The y axis is a log₂ scale. M is defined as the log₂ (intensity₁/intensity₂). Three comparisons were repeated for the breast tumors and four comparisons for the colon tumors.

Figure 3

A: Hierarchical clustering for the 52 breast samples (26 snap-frozen and 26 RNAlater preservative-suspended). B: Hierarchical clustering for the 44 colon samples (22 snap-frozen and 22 RNAlater preservative-suspended, including repeats). Each column represents one sample, and each row represents one gene. The sample type (RNAlater-preserved or snap-frozen) is coded by color and by the letter R (for RNAlater-preserved) or T (for snap-frozen).