Evaluation and validation of total RNA extraction methods for microRNA expression analyses in formalin-fixed, paraffin-embedded tissues

Abstract

Histopathology archives of well-annotated formalin-fixed, paraffin-embedded (FFPE) tissue specimens are valuable resources for retrospective studies of human diseases. Since recovery of quality intact mRNA compatible with molecular techniques is often difficult due to degradation, we evaluated microRNA (miRNA), a novel class of small RNA molecules with growing therapeutic and diagnostic potential, as an alternative analyte for gene expression studies of FFPE samples. Analyzing total RNA yield, miRNA recovery, and robustness of real-time polymerase chain reaction for miRNA, mRNA, and rRNA species, we compared the performance of commercially available RNA isolation kits and identified a preferred methodology. We further implemented modifications to increase tissue throughput and incorporate a quantitative Armored RNA process control to monitor RNA recovery efficiency. The optimized process was tested for reproducibility as well as interoperator and interday variability, and was validated with a set of 30 clinical samples. In addition, we demonstrated that, independent of FFPE block age and RNA quality, miRNAs generate quantitative reverse transcription-polymerase chain reaction signals that are more robust and better correlate with expression levels from frozen reference samples compared with longer mRNAs. Our broad study, including a total of 272 independent RNA isolations from 17 tissue types and 65 FFPE blocks up to 12 years old, indicates that miRNAs are not only suitable but are also likely superior analytes for the molecular characterization of compromised archived clinical specimens.

Figure 1

qRT-PCR analysis of RNA samples isolated with the RecoverAll or RNeasy kit. Total RNA was extracted in triplicate from five different FFPE tissue blocks using the indicated method. Each RNA sample was analyzed in duplicate using qRT-PCR assays specific for miR-24, -103, -191, 18S rRNA, or hGUSB mRNA.

Figure 2

Comparison between FFPE and matching frozen samples. A: Average expression levels for miR-24, -103, and -191 in RNA samples isolated from FFPE and matched frozen breast (three specimens), cervix (three specimens), and gall bladder (two specimens) tissues. Each qRT-PCR reaction was performed in duplicate. B: Same as A for 18S rRNA and hGUSB mRNA. C: Average differential expression (ΔCt) between frozen and matching FFPE samples for miRNAs or mRNA and rRNA species. D: Average standard deviation of qRT-PCR signal (Ct) within the 13 FFPE tissues blocks described in Table 1 for miRNA or mRNA and rRNA species.

Figure 3

Scale-up of the RecoverAll procedure. RNA isolation was performed with four, eight, 12, or 16 FFPE tissue slices (20-μm) from two prostate cancer FFPE blocks that differed in tissue cross-sectional area. For the block with a tissue area >150 mm², twice the recommended amount of proteinase K was used.

Figure 4

Armored RNA Quant as a process control. RNA isolation was performed using 16 20-μm slices from 15 pairs of matched FFPE lung tumor and NAT specimens with 10¹⁰ copies of ARQ spiked at the proteinase K digestion step. qRT-PCR were performed in duplicate using 2 μl of purified RNA for ARQ and 10 ng of purified RNA for miR-24, -191, -103, hGUSB mRNA, and 18S rRNA. Samples with abnormally high Ct for endogenous RNAs and ARQ or for endogenous RNAs only are indicated by a circle and a star, respectively.