No abstract

A quantitative polymerase chain reaction technique (qPCR) in combination with scanning electron microscopy was applied for the evaluation of early gene expression response and cellular reactions close to titanium implants. Anodically oxidized and machined titanium miniscrews were inserted in rat tibiae. After 1, 3, and 6 days the implants were unscrewed and the surrounding bone was retrieved using trephines. Both the implants and bone were analyzed with qPCR. A greater amount of cells, as indicated with higher expression of 18S, was detected on the oxidized surface after 1 and 6 days. Significantly higher osteocalcin (at day 6), alkaline phosphatase (at days 3 and 6), and cathepsin K (at day 3) expression was demonstrated for the oxidized surface. Higher expression of tumor necrosis factor-alpha (at day 1) and interleukin-1beta (at days 1 and 6) was detected on the machined surfaces. SEM revealed a higher amount of mesenchymal-like cells on the oxidized surface. The results show that the rapid recruitment of mesenchymal cells, the rapid triggering of gene expression crucial for bone remodeling and the transient nature of inflammation, constitute biological mechanisms for osseointegration, and high implant stability associated with anodically oxidized implants.

There is a general lack of rapid, sensitive, and quantitative methods for the detection of differentiating human embryonic stem cells (hESCs). Using light microscopy and immunohistochemistry, we observed that morphological changes of differentiating hESCs precede any major alterations in the expression of several commonly used hESC markers (SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and Nanog). In an attempt to quantify the changes during stochastic differentiation of hESCs, we developed a robust and sensitive multimarker quantitative real-time polymerase chain reaction (QPCR) method. To maximize the sensitivity of the method, we measured the expression of up-and downregulated genes before and after differentiation of the hESCs. Out of the 12 genes assayed, we found it clearly sufficient to determine the relative differentiation state of the cells by calculating a collective expression index based on the mRNA levels of Oct-4, Nanog, Cripto, and α-fetoprotein. We evaluated the method using different hESC lines maintained in either feeder-dependent or feeder-free culture conditions. The QPCR method is very flexible, and by appropriately selecting reporter genes, the method can be designed for various applications. The combination of QPCR with hESC-based technologies opens novel avenues for high-throughput analysis of hESCs in, for example, pharmacological and cytotoxicity screening. Stem

Advances in the biologic sciences and technology are providing molecular targets for diagnosis and treatment of cancer. Lymphoma is a group of cancers with diverse clinical courses. Gene profiling opens new possibilities to classify the disease into subtypes and guide a differentiated treatment. Real-time PCR is characterized by high sensitivity, excellent precision and large dynamic range, and has become the method of choice for quantitative gene expression measurements. For accurate gene expression profiling by real-time PCR, several parameters must be considered and carefully validated. These include the use of reference genes and compensation for PCR inhibition in data normalization. Quantification by real-time PCR may be performed as either absolute measurements using an external standard, or as relative measurements, comparing the expression of a reporter gene with that of a presumed constantly expressed reference gene. Sometimes it is possible to compare expression of reporter genes only, which improves the accuracy of prediction. The amount of biologic material required for real-time PCR analysis is much lower than that required for analysis by traditional methods due to the very high sensitivity of PCR. Fine-needle aspirates and even single cells contain enough material for accurate real-time PCR analysis.

Currently, in real-time PCR, one often has to choose between using a sequence-specific probe and a nonspecific double-stranded DNA (dsDNA) binding dye for the detection of amplified DNA products. The sequence-specific probe has the advantage that it only detects the targeted product, while the nonspecific dye has the advantage that melting curve analysis can be performed after completed amplification, which reveals what kind of products have been formed. Here we present a new strategy based on combining a sequence-specific probe and a nonspecific dye, BOXTO, in the same reaction, to take the advantage of both chemistries. We show that BOXTO can be used together with both TaqMan probes and locked nucleic acid (LNA) probes without interfering with the PCR. The probe signal reflect formation of target product, while melting curve analysis of the BOXTO signal reveals primer-dimer formation and the presence of any other anomalous products.