Proper normalization is a critical but often an underappreciated aspect of quantitative gene expression analysis. This study describes the identification and characterization of appropriate reference RNA targets for the normalization of microRNA (miRNA) quantitative RT-PCR data. miRNA microarray data from dozens of normal and disease human tissues revealed ubiquitous and stably expressed normalization candidates for evaluation by qRT-PCR. miR-191 and miR-103, among others, were found to be highly consistent in their expression across 13 normal tissues and five pair of distinct tumor/normal adjacent tissues. These miRNAs were statistically superior to the most commonly used reference RNAs used in miRNA qRT-PCR experiments, such as 5S rRNA, U6 snRNA, or total RNA. The most stable normalizers were also highly conserved across flashfrozen and formalin-fixed paraffin-embedded lung cancer tumor/NAT sample sets, resulting in the confirmation of one welldocumented oncomir (let-7a), as well as the identification of novel oncomirs. These findings constitute the first report describing the rigorous normalization of miRNA qRT-PCR data and have important implications for proper experimental design and accurate data interpretation.

BACKGROUND Fragile X Syndrome (FXS) is a trinucleotide repeat disease that is caused by the expansion of CGG sequences in the 5’ untranslated region of the FMR1 gene. Molecular diagnoses of FXS and other emerging FMR1 disorders typically rely on two tests, PCR and Southern blotting. However, performance or throughput limitations in these methods currently constrain routine testing. METHODS We evaluated a novel FMR1 gene-specific PCR technology with 20 cell line DNA templates and 146 blinded clinical specimens. The CGG repeat number was determined by fragment sizing of PCR amplicons using capillary electrophoresis and compared with the results of FMR1 Southern blotting performed with the same samples. RESULTS The FMR1 PCR accurately detected full mutation alleles up to at least 1300 CGG repeats and comprising >99% GC character. All categories of alleles detected by Southern blot, including 66 specimens with full mutations, were also identified by FMR1 PCR for each of 146 clinical specimens. Since all full mutation alleles in heterozygous female samples were detected by PCR, allele zygosity was reconciled in every case. The PCR reagents also detected a 1% mass fraction of a 940 CGG allele in a background of 99% 23 CGG allele—roughly 5-fold greater sensitivity than Southern blotting. CONCLUSIONS The novel PCR technology can accurately categorize the spectrum of FMR1 alleles, including alleles previously considered too large to amplify, reproducibly detect low abundance full mutation alleles, and correctly infer homozygosity in female specimens, thus greatly reducing the need for sample reflexing to Southern blot.

NOTCH1 mutations have been reported to occur in 10 to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation and mutation analyses. Copy number increases were identified in NOTCH pathway genes including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4/37 of the tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptors mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently.

(CGG) n repeat expansion in the FMR1 gene is associated with fragile X syndrome and other disorders. Current methods for FMR1 molecular testing rely on Southern blot analysis to detect expanded alleles too large to be PCR-amplified and to identify female homozygous alleles that often confound interpretations of PCR data. A novel , single-tube CGG repeat primed FMR1 PCR technology was designed with two genespecific primers that flank the triplet repeat region, as well as a third primer that is complementary to the (CGG) n repeat. This PCR was evaluated with 171 unique DNA samples , including a blinded set of 146 clinical specimens. The method detected all alleles reported by Southern blot analysis , including full mutations in 66 clinical samples and comprised up to 1300 CGG. Furthermore , a blinded cohort of 42 female homozygous and heterozygous specimens, including 21 with full mutation alleles , was resolved with 100% accuracy. Last , AGG interrupter sequences, which may influence the risk of (CGG) n expansion in the children of some carriers , were each correctly identified in 14 male and female clinical samples as referenced to DNA sequencing. As a result , this PCR provides robust detection of expanded alleles and resolves allele zygosity , thus minimizing the number of samples that require Southern blot analysis and producing more comprehensive FMR1 genotyping data than other methods. Expansion of cytosine-guanine-guanine (CGG) triplet repeats in the 5Ј-untranslated region of the fragile X mental retardation 1 (FMR1, NM_002024.4) gene is associated with several disorders, including fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X-associated primary ovarian insufficiency. [1][2][3][4] Patients with the FMR1 full mutation (Ͼ200 CGG repeats) may be affected by a range of neurological, psychiatric, or emotional challenges, including mental retardation and/or autism.5 Deficits in development and particularly in attention and social communication have also been noted for many children with the FMR1 premutation. Moreover, premutation carriers (55 to 200 CGG repeats) are known to be at risk for fragile X-associated primary ovarian insufficiency and fragile X-associated tremor/ataxia syndrome, and some of these individuals may present additional complications, such as hypothyroidism and fibromyalgia.6 As a result, FMR1 disorders are linked to a range of clinical conditions, necessitating testing patients at different times during their life span. 7Fragile X syndrome molecular diagnosis is usually based on quantification of the (CGG) n repeat elements and the assessment of the methylation state of expanded alleles.5 Although PCR is the preferred approach to determine the (CGG) n repeat length of FMR1 alleles, typically only alleles with less than 100 to 150 CGG have

Implementation of highly sophisticated technologies, such as next-generation sequencing (NGS), into routine clinical practice requires compatibility with common tumor biopsy types, such as formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration specimens, and validation metrics for platforms, controls, and data analysis pipelines. In this study, a two-step PCR enrichment workflow was used to assess 540 known cancer-relevant variants in 16 oncogenes for high-depth sequencing in tumor samples on either mature (Illumina GAIIx) or emerging (Ion Torrent PGM) NGS platforms. The results revealed that the background noise of variant detection was elevated approximately twofold in FFPE compared with cell line DNA. Bioinformatic algorithms were optimized to accommodate this background. Variant calls from 38 residual clinical colorectal cancer FFPE specimens and 10 thyroid fine-needle aspiration specimens were compared across multiple cancer genes, resulting in an accuracy of 96.1% (95% CI, 96.1% to 99.3%) compared with Sanger sequencing, and 99.6% (95% CI, 97.9% to 99.9%) compared with an alternative method with an analytical sensitivity of 1% mutation detection. A total of 45 of 48 samples were concordant between NGS platforms across all matched regions, with the three discordant calls each represented at <10% of reads. Consequently, NGS of targeted oncogenes in real-life tumor specimens using distinct platforms addresses unmet needs for unbiased and highly sensitive mutation detection and can accelerate both basic and clinical cancer research.