Evaluation of microRNA expression in patient bone marrow aspirate slides

Abstract

Like formalin fixed paraffin embedded (FFPE) tissues, archived bone marrow aspirate slides are an abundant and untapped resource of biospecimens that could enable retrospective molecular studies of disease. Historically, RNA obtained from slides is limited in utility because of their low quality and highly fragmented nature. MicroRNAs are small (≈ 22 nt) non-coding RNA that regulate gene expression, and are speculated to preserve well in FFPE tissue. Here we investigate the use of archived bone marrow aspirate slides for miRNA expression analysis in paediatric leukaemia. After determining the optimal method of miRNA extraction, we used TaqMan qRT-PCR to identify reference miRNA for normalisation of other miRNA species. We found hsa-miR-16 and hsa-miR-26b to be the most stably expressed between lymphoblastoid cell lines, primary bone marrow aspirates and archived samples. We found the average fold change in expression of hsa-miR-26b and two miRNA reportedly dysregulated in leukaemia (hsa-miR-128a, hsa-miR-223) was <0.5 between matching archived slide and bone marrow aspirates. Differential expression of hsa-miR-128a and hsa-miR-223 was observed between leukaemic and non-leukaemic bone marrow from archived slides or flash frozen bone marrow. The demonstration that archived bone marrow aspirate slides can be utilized for miRNA expression studies offers tremendous potential for future investigations into the role miRNA play in the development and long term outcome of hematologic, as well as non-hematologic, diseases.

Figure 1. The stability of miRNA species expression in hematopoietic samples.

The stability of miRNA species expression using 41 samples of leukaemic and non-leukaemic cell lines, paediatric archived bone marrow aspirate slides (Table S1), and 14 potential miRNA candidates (Table S2). geNorm and NormFinder (NF) were used to calculate the expression stability values for each miRNA tested. The most ‘stably’ expressed gene in a sample group is that with an average expression stability value (M) closest to zero. The most stably expressed miRNA were; NF Ungrouped: hsa-miR-16 M = 1.143; NF Grouped by Cancer Status: hsa-miR-16 M = 0.457; NF Grouped by Leukaemia subtype: hsa-miR-16 M = 0.573; geNorm: hsa-miR-16 and hsa-miR-26b are equal at M = 1.817.

Figure 2. A comparison of RNA extraction methods for the evaluation of RNA concentration (ng/µl) and miRNA expression from archived bone marrow aspirate slides.

The comparison of miRNA extraction methods from matched unstained archived bone marrow smears (n = 6 or n = 8 patients; Table S3). Six different methods were used based on traditional TRIzol or using the Roche High Pure miRNA Column extraction kit (Table 1). (A) Comparison of Total RNA yield from experimental extraction protocols. The average RNA concentration was measured by NanoDrop (ng/µl ±s.e.m.). Kit Total RNA extraction methods with a Proteinase K digestion (‘FFPE protocol’) are higher in RNA yield compared to kit methods without a digestion step (‘Tissue protocol’), or when extracting small RNA only. These methods are also significantly better than TRIzol extractions. The archived slide extraction method with the highest RNA yield was the Roche kit ‘isolation from FFPE Tissue’ Total RNA extraction with an overnight Proteinase K digestion. * Indicates RNA concentration is significantly different to the Kit Overnight FFPE: Total RNA protocol. (B) Comparison of hsa-miR-16 expression from experimental extraction protocols. The average expression of our reverence gene (hsa-miR-16) measured by qRT-PCR (raw C_t ±s.e.m.). Raw C_t value tracks with total RNA yield, whereby as the concentration of RNA increases the C_t value decreases. This analysis shows extractions with a Proteinase K digestion return a lower C_t value compared to extractions without a digestion step. TRIzol extractions also return a higher C_t value than the Proteinase K digestion protocols.

Figure 3. The average fold change in miRNA expression between matched patient fresh bone marrow aspirates and archived bone marrow slides.

The average fold change in miRNA expression between matching time-point patient fresh bone marrow aspirates and archived slide bone marrow aspirates (Table S4). (A) microRNA expression of matched patient fresh bone marrow and archived slide samples. Three miRNA TaqMan assays were used (hsa-miR-128a, hsa-miR-26b, hsa-miR-223) and the Livak method for Fold Change analysis to analyse the fold change difference between fresh Bone marrow and matching archived slides. hsa-miR-128a and hsa-miR-223 have been previously reported to have altered expression in leukaemia, whereas we have found here hsa-miR-26b is a stable reference. The overall difference in miRNA expression between fresh bone marrow samples and their matching archived slide as shown here is negligible. (B) Difference in hsa-miR-223 expression in archived and fresh leukaemic samples. Previously reported dysregulated miRNA hsa-miR-223 was used to analyse the fold change difference between leukaemic and non-leukaemic samples, to gauge the biological relevance of archived specimens in comparison to fresh. All samples were normalized to hsa-miR-26b. AML and ALL both showed fold change differences of hsa-miR-223 compared to non-leukaemic samples. The bone marrow and archived slides showed similar expression in both AML (Bone Marrow: 5.1±1.99 s.e.m.; Slide: 4.4±1.94 s.e.m.) and ALL (Bone Marrow: 2.71±0.81 s.e.m.; Slide: 2.1±0.83 s.e.m.).