A functional assay for microRNA target identification and validation

MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3′-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets

Blood First Edition paper

Key Points • There is 100% concordance in the cytogenetic and mutation profile between PB and BM in myelodysplastic syndrome. Recent studies have shown that more than 80% of bone marrow (BM) samples from patients with myelodysplastic syndrome (MDS) harbor somatic mutations and/or genomic aberrations, which are of diagnostic and prognostic importance. We investigated the potential use of peripheral blood (PB) and serum to identify and monitor BM-derived genetic markers using high-resolution single nucleotide polymorphism array (SNP-A) karyotyping and parallel sequencing of 22 genes frequently mutated in MDS. This pilot study showed a 100% SNP-A karyotype concordance and a 97% mutation concordance between the BM and PB. In contrast, mutation analysis using Sanger sequencing of PB and serum-derived DNA showed only 65% and 42% concordance to BM, respectively. Our results show the potential utility of PB as a surrogate for BM for MDS patients, thus avoiding the need for repeated BM aspirates particularly in elderly patients and those with fibrotic or hypocellular marrows. (Blood. 2013;122(4):567-570) Introduction The myelodysplastic syndromes (MDSs) are clonal disorders of hematopoiesis that occur predominantly in the elderly (median age 72 years) and are characterized by morphologic dysplasia, ineffective hematopoiesis, peripheral blood (PB) cytopenias, chromosomal aberrations, and propensity to myeloid leukemic transformation. The advent of high-throughput and high-resolution techniques for genetic analysis has shown that more than 80% of MDS patients harbor somatic mutations and/or genomic aberrations in their bone marrow (BM), which provide pathogenetic as well as diagnostic and prognostic insights into this disease. 1-4 Frequent BM aspirates may be required for morphological Study design Genomic DNA from PB and BM was extracted (Qiagen) from frozen cell pellets and 100 ng was whole genome amplified (WGA; Qiagen), both per manufacturer's protocols. Serum DNA was purified from 200 mL of serum using a modified sodium iodide/Triton-based lysis followed by isopropanol precipitation as described. 12 Affymetrix SNP 6.0 array (SNP-A) karyotyping and 454-PS of all exons of DNMT3a, RUNX1, CEBPa, TP53, EZH2, and ZRSR2 and mutation "hot spots" for NPM1, FLT3, ASXL1, IDH1, IDH2, MPL, JAK2, BRAF, cCBL, NRAS, KRAS, C-KIT, SF3B1, SRSF2, and U2AF35 were performed and analyzed as previously described. 13,14 TET2 was analyzed using Sanger sequencing. Independent validation for all mutations was performed using Sanger sequencing of unamplified genomic DNA. Polymerase chain reaction (PCR) conditions for serum were identical to those for PB; however, a second 10-cycle PCR reaction using nested primers (US1-GTAGTGCGATGGCCAGT, US2-CAGTGTGCAGCGATGAC) was required to provide adequate amplicon yield for Sanger sequencing. The study was approved by the local research ethics committee under project 0033 and conducted in accordance with the Declaration of Helsinki. Results and discussion Karyotype analysis Karyotype aberrations were assessed using SNP-A on PB samples from 31 MDS patients, from whom metaphase cytogenetics (MC) and BM SNP-A karyotypes were available. These consisted of th

Utility of peripheral blood for cytogenetic and mutation analysis in myelodysplastic syndrome

Key Points There is 100% concordance in the cytogenetic and mutation profile between PB and BM in myelodysplastic syndrome.</jats:p