Universal nucleic acid sequence-based amplification for simultaneous amplification of messengerRNAs and microRNAs.

A universal NASBA assay is presented for simultaneous amplification of multiple microRNA (miRNA) and messengerRNA (mRNA) sequences. First, miRNA and mRNA sequences are reverse transcribed using tailed reverse transcription primer pairs containing a gene-specific and an non-specific region. For reverse transcription of small miRNA molecules a non-specific region is incorporated into a structured stem-loop reverse transcription primer. Second, a universal NASBA primer pair that recognizes the tagged cDNA molecules enables a simultaneous, transcription-based amplification reaction (NASBA) of all different cDNA molecules in one reaction. The NASBA products (RNA copies) are detected by gene-specific DNA probes immobilized on a biochip. By using the multiplex reverse transcription combined with the universal NASBA amplification up to 14 different mRNA and miRNA sequences can be specifically amplified and detected in parallel. In comparison with standard multiplex NASBA assays this approach strongly enhances the multiplex capacity of NASBA-based amplification reactions. Furthermore simultaneous assaying of different RNA classes can be achieved that might be beneficial for studying miRNA-based regulation of gene expression or for RNA-based tumor diagnostics.

Nucleic acid sequence-based amplification in formalin-fixed and paraffin-embedded breast-cancer tissues.

AIM: To evaluate the nucleic acid sequence-based amplification (NASBA) technique to amplify mRNA isolated from formalin-fixed and paraffin-embedded (FFPE) breast-cancer tissues. METHODS: RNA was extracted from archived, 10-year-old FFPE tissues, and selected genes, namely ribosomal protein S18 (RPS18), epidermal growth factor receptor 2 (HER2), estrogen receptor alpha (ERα), Y box binding protein (YBX-1), matrix metallopeptidase 11 (MMP11), caspase 8 (CASP8) and superoxide dismutase 2 (SOD2), were amplified by NASBA. RESULTS: Despite strong degradation of the template, RNA amplification of all tested genes resulted in strong hybridisation signals. Sensitivity tests showed that the RPS18 NASBA assay was more sensitive than real-time RT-PCR used as a reference method. The sensitivity of the HER2, ERα, MMP11, YBX1, CASP8 and SOD2 NASBA assay was comparable with RT-PCR targeted to the respective genes. CONCLUSIONS: The results obtained indicate that NASBA is suitable to amplify with high specificity and sensitivity, even strongly degraded RNA isolated from FFPE tissues, and therefore can complement already-existing amplification techniques such as RT-PCR for analysis of such tissues.

Microarray-based amplification and detection of RNA by nucleic acid sequence based amplification.

Nucleic acid sequence based amplification (NASBA) is a versatile in vitro nucleic acid amplification method. In this work, RNA amplification and labeling by NASBA and microarray analysis are combined in a one-step process. The NASBA reaction is performed in direct contact with capture probes. These probes are bound to surface-attached hydrogel spots generated at the chip surfaces by using a simple printing and UV irradiation process. Five gene expression and SNP parameters with known relevance in breast cancer diagnostics were chosen to demonstrate that multiplex NASBA-on-microarray analysis is possible. A minimum amount of 10 pg of total RNA was shown to be sufficient for the detection of the reference parameter RPS18, which demonstrates that the detection limit of the microarray-based NASBA assays theoretically allows single-cell assays to be performed.

Effects of phosphodiesterase 7 inhibition by RNA interference on the gene expression and differentiation of human mesenchymal stem cell-derived osteoblasts.

The second messenger molecule cyclic adenosine monophosphate (cAMP) plays an important role in the hormonal regulation of bone metabolism. cAMP is inactivated by the cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 known families designated PDE 1-11. The aim of this study was to investigate the effect of PDE7 and PDE8 inhibition on the gene expression and differentiation of human osteoblasts. Osteoblasts differentiated from human mesenchymal stem cells (hMSC) were cultured and treated with short interfering RNAs (siRNAs) generated from PDE7 and PDE8 PCR products. Total RNA was isolated from the cells, and gene expression was assayed with cDNA microarray and quantitative real-time PCR. bALP measurements were assayed during differentiation, and mineralization was determined by quantitative Alizarin red S staining. PDE7 and PDE8 inhibition by RNA interference decreased the gene expression of PDE7A by 60-70%, PDE7B by 40-50%, and PDE8A by 30%. PDE7 silencing increased the expression of beta-catenin, osteocalcin, caspase-8, and cAMP-responsive element-binding protein 5 (CREB-5) genes and decreased the expression of the 1, 25-dihydroxyvitamin D3 receptor gene. PDE8A silencing increased the expression of anti-apoptotic genes, but decreased the expression of osteoglycin (osteoinductive factor) and bone morphogenetic protein 1 (BMP-1). PDE7 silencing increased bALP and mineralization up to three-fold compared to controls. Treatment with the PDE7-selective PDE inhibitor BRL-50481 had similar effects on mineralization as the gene silencing. The PDE7 silencing also increased forskolin stimulated cAMP response, but had no effect on the proliferation rate. Furthermore, osteocalcin expression was increased by PDE7 silencing by a mechanism dependent on protein kinase A. Our results show that specific gene silencing with the RNAi method is a useful tool for inhibiting the gene expression of specific PDEs and that PDE7 silencing upregulates several osteogenic genes and increases mineralization. PDE7 may play an important role in the regulation of osteoblastic differentiation.

Extracellular calcium regulates parathyroid hormone-related peptide expression in osteoblasts and osteoblast progenitor cells.

Parathyroid hormone-related peptide (PTHrP) has been shown to have anabolic effects on bone in women with postmenopausal osteoporosis. On the cellular level PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The calcium concentration is considerably higher in the vicinity of resorbing osteoclasts than in the plasma. Therefore the osteoblasts are likely to be confronted by elevated extracellular calcium concentrations in the areas of resorptive activity. The present study was designed to assess the possibility that extracellular calcium could regulate PTHrP expression in osteoblastic cells. Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The PTHrP release into the culture media was measured by an immunoradiometric assay and the expression of PTHrP, osteocalcin and Runx2 mRNA was assayed by real-time PCR. Increasing the extracellular calcium from 1 mM to 5 mM for 24 h resulted in a 4-6-fold increase in the PTHrP release. PTHrP mRNA was also increased by elevated calcium levels. The effect of calcium stimulation on PTHrP release could be seen within 60 min of treatment. The extracellular calcium sensing receptor (CaR) agonist neomycin mimicked the effects of calcium and the MEK/MAPK inhibitor PD98059 abolished the effect of calcium and neomycin. High extracellular calcium increased the mineralization of hMSC and the expression of osteocalcin, but this effect was not mimicked by neomycin. Our results show that in hMSC, elevated extracellular calcium levels increases both released PTHrP and PTHrP mRNA expression. The effect of calcium on PTHrP can be mimicked by activation of the CaR and can be diminished by inhibition of the MAPK signalling pathway.