Multi-platform analysis of microRNA expression measurements in RNA from fresh frozen and FFPE tissues.

MicroRNAs play a role in regulating diverse biological processes and have considerable utility as molecular markers for diagnosis and monitoring of human disease. Several technologies are available commercially for measuring microRNA expression. However, cross-platform comparisons do not necessarily correlate well, making it difficult to determine which platform most closely represents the true microRNA expression level in a tissue. To address this issue, we have analyzed RNA derived from cell lines, as well as fresh frozen and formalin-fixed paraffin embedded tissues, using Affymetrix, Agilent, and Illumina microRNA arrays, NanoString counting, and Illumina Next Generation Sequencing. We compared the performance within- and between the different platforms, and then verified these results with those of quantitative PCR data. Our results demonstrate that the within-platform reproducibility for each method is consistently high and although the gene expression profiles from each platform show unique traits, comparison of genes that were commonly detectable showed that detection of microRNA transcripts was similar across multiple platforms.

Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays.

BACKGROUND: MicroRNAs (miRNAs) represent a growing class of small non-coding RNAs that are important regulators of gene expression in both plants and animals. Studies have shown that miRNAs play a critical role in human cancer and they can influence the level of cell proliferation and apoptosis by modulating gene expression. Currently, methods for the detection and measurement of miRNA expression include small and moderate-throughput technologies, such as standard quantitative PCR and microarray based analysis. However, these methods have several limitations when used in large clinical studies where a high-throughput and highly quantitative technology needed for the efficient characterization of a large number of miRNA transcripts in clinical samples. Furthermore, archival formalin fixed, paraffin embedded (FFPE) samples are increasingly becoming the primary resource for gene expression studies because fresh frozen (FF) samples are often difficult to obtain and requires special storage conditions. In this study, we evaluated the miRNA expression levels in FFPE and FF samples as well as several lung cancer cell lines employing a high throughput qPCR-based microfluidic technology. The results were compared to standard qPCR and hybridization-based microarray platforms using the same samples. RESULTS: We demonstrated highly correlated Ct values between multiplex and singleplex RT reactions in standard qPCR assays for miRNA expression using total RNA from A549 (R = 0.98; p < 0.0001) and H1299 (R = 0.95; p < 0.0001) lung cancer cell lines. The Ct values generated by the microfluidic technology (Fluidigm 48.48 dynamic array systems) resulted in a left-shift toward lower Ct values compared to those observed by ABI 7900 HT (mean difference, 3.79), suggesting that the microfluidic technology exhibited a greater sensitivity. In addition, we show that as little as 10 ng total RNA can be used to reliably detect all 48 or 96 tested miRNAs using a 96-multiplexing RT reaction in both FFPE and FF samples. Finally, we compared miRNA expression measurements in both FFPE and FF samples by qPCR using the 96.96 dynamic array and Affymetrix microarrays. Fold change comparisons for comparable genes between the two platforms indicated that the overall correlation was R = 0.60. The maximum fold change detected by the Affymetrix microarray was 3.5 compared to 13 by the 96.96 dynamic array. CONCLUSION: The qPCR-array based microfluidic dynamic array platform can be used in conjunction with multiplexed RT reactions for miRNA gene expression profiling. We showed that this approach is highly reproducible and the results correlate closely with the existing singleplex qPCR platform at a throughput that is 5 to 20 times higher and a sample and reagent usage that was approximately 50-100 times lower than conventional assays. We established optimal conditions for using the Fluidigm microfluidic technology for rapid, cost effective, and customizable arrays for miRNA expression profiling and validation.

Ret-PCP2 colocalizes with protein kinase C in a subset of primate ON cone bipolar cells.

Purkinje cell protein 2 (PCP2), a member of the family of guanine dissociation inhibitors and a strong interactor with the G-protein subunit G alpha(o), localizes to retinal ON bipolar cells. The retina-specific splice variant of PCP2, Ret-PCP2, accelerates the light response of rod bipolar cells by modulating the mGluR6 transduction cascade. All ON cone bipolar cells express mGluR6 and G alpha(o), but only a subset expresses Ret-PCP2. Here we test the hypothesis that Ret-PCP2 contributes to shaping the various temporal bandwidths of ON cone bipolar cells in monkey retina. We found that the retinal splice variants in monkey and mouse are similar and longer than the cerebellar variants. Ret-PCP2 is strongly expressed by diffuse cone bipolar type 4 cells (DB4; marked with anti-PKCalpha) and weakly expressed by midget bipolar dendrites (labeled by antibodies against G alpha(o), G gamma 13, or mGluR6). Ret-PCP2 is absent from diffuse cone bipolar type 6 (DB6; marked with anti-CD15) and blue cone bipolar cells (marked with anti-CCK precursor). Thus, cone bipolar cells that terminate in stratum 3 of the inner plexiform layer (DB4) express more Ret-PCP2 than those that terminate in strata 3 + 4 (midget bipolar cells), and these in turn express more than those that terminate in stratum 5 (DB6 and blue cone bipolar cells). This expression pattern approximates the arborization of ganglion cells (GC) with different temporal bandwidths: parasol GCs stratifying near stratum 3 are faster than midget GCs stratifying in strata 3 + 4, and these are probably faster than the sluggish GCs that arborize in stratum 5.

Retinal ON bipolar cells express a new PCP2 splice variant that accelerates the light response.

PCP2, a member of the GoLoco domain-containing family, is present exclusively in cerebellar Purkinje cells and retinal ON bipolar cells. Its function in these tissues is unknown. Biochemical and expression system studies suggest that PCP2 is a guanine nucleotide dissociation inhibitor, although a guanine nucleotide exchange factor has also been suggested. Here, we studied the function of PCP2 in ON bipolar cells because their light response depends on Galpha(o1), which is known to interact with PCP2. We identified a new splice variant of PCP2 (Ret-PCP2) and localized it to rod bipolar and ON cone bipolar cells. Electroretinogram recordings from PCP2-null mice showed a normal a-wave but a slower falling phase of the b-wave (generated by the activity of ON bipolar cells) relative to the wild type. Whole-cell recordings from rod bipolar cells showed, both under Ames medium and after blocking GABA(A/C) and glycine receptors, that PCP2-null rod bipolar cells were more depolarized than wild-type cells with greater inward current when clamped to -60 mV. Also under both conditions, the rise time of the response to intense light was slower by 28% (Ames) and 44% (inhibitory blockers) in the null cells. Under Ames medium, we also observed >30% longer decay time in the PCP2-null rod bipolar cells. We conclude that PCP2 facilitates cation channels closure in the dark, shortens the rise time of the light response directly, and accelerates the decay time indirectly via the inhibitory network. These data can most easily be explained if PCP2 serves as a guanine nucleotide exchange factor.

Gli1 induces G2/M arrest and apoptosis in hippocampal but not tumor-derived neural stem cells.

Sonic hedgehog (Shh) is necessary for sustaining the proliferation of neural stem cells (NSCs), yet little is known about its mechanisms. Whereas Gli1, Gli2, and Gli3, the primary mediators of Shh signaling, were all expressed in hippocampal neural progenitors, Shh treatment of NSCs induced only Gli1 expression. Acute depletion of Gli1 in postnatal NSCs by short-hairpin RNA decreased proliferation, whereas germline deletion of Gli1 did not affect NSC proliferation, suggesting a difference in mechanisms of Gli1 compensation that may be developmentally dependent. To determine whether Gli1 was sufficient to enhance NSC proliferation, we overexpressed this mitogen and were surprised to find that Gli1 resulted in decreased proliferation, accumulation of NSCs in the G2/M phase of cell cycle, and apoptosis. In contrast, Gli1-expressing lineage-restricted neural precursors demonstrated a 4.5-fold proliferation enhancement. Expression analyses of Gli1-expressing NSCs identified significant induction of Gadd45a and decreased cyclin A2 and Stag1 mRNA, genes involved in the G2-M transition and apoptosis. Furthermore, Gadd45a overexpression was sufficient to partially recapitulate the Gli1-induced G2/M accumulation and cell death of NSCs. In contrast to normal stem cells, tumor-derived stem cells had markedly higher basal Gli1 expression and did not undergo apoptosis with further elevation of Gli1. Our data suggest that Gli1-induced apoptosis may serve as a protective mechanism against premature mitosis and may give insight into mechanisms by which nonmalignant stem cells restrain hyperproliferation in the context of potentially transforming mitogenic signals. Tumor-derived stem cells apparently lack these mechanisms, which may contribute to their unrestrained proliferation and malignant potential.

Enhanced Purkinje cell survival but compromised cerebellar function in targeted anti-apoptotic protein transgenic mice.

Regulation of Purkinje cell (PC) number is critical for proper assembly and function of the cerebellum. Murine cerebellar neurogenesis yields supernumerary populations of cells that are subject to programmed cell death during development and aging. This study focuses on the control of mouse PC number during development and the consequences of interrupting normal cell death. Purkinje cell-specific regulatory elements from the pcp2 gene were employed to target expression of two anti-apoptotic proteins, human BCL-2 and adenovirus E1B 19k to the PCs of transgenic mice. Comparative morphometric analyses indicated no significant difference in PC numbers in the strongest BCL-2 expressing line, while a 14.2% increase was noted in the pcp2/E1B 19k transgenic line. The temporal transgene expression patterns of several mouse lines indicated that PC numbers are normally adjusted during the first postnatal week. Crossbreeding studies demonstrated that both Bcl-2 and E1B 19k transgenes provided Purkinje cell protection from SV40 Tag-induced cell death. Interestingly, RotaRod behavioral analysis demonstrated that 'rescued' Purkinje cells degrade cerebellar function. Furthermore, aged E1B 19k and Bcl-2 mice exhibited decreased RotaRod performance despite increased PC numbers. These findings have implications regarding neuronal death during development and aging as well as cellular and genetic strategies to circumvent neuronal degeneration.