Functional siRNA expression from transfected PCR products

Cloning vectors for expression-PCR products.

Manufacturing DNA microarrays from unpurified PCR products.

For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coa...

Long-term suppression of telomerase expression in HeLa cell clones, transfected with an expression vector carrying siRNA targeting hTERT mRNA.

HeLa cell cultures were used as model systems for small interfering RNA (siRNA) induced knockdown of mRNA expression of the human telomerase catalytic subunit, telomerase reverse transcriptase (hTERT). Four 21-bp siRNAs targeting different sites of the hTERT mRNA were designed, and the siRNA molecules produced by a T7 transcription system in vitro. In transient transfection assays on HeLa cells...

Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing

Chitosan, a well known natural cationic polysaccharide, has been successfully implemented in vitro and in vivo as a nonviral delivery system for both plasmid DNA and siRNA. While using chitosan/siRNA polyplexes to knock down specific targets, we have underestimated the effect of nucleic acids binding to chitosan when extracting RNA for subsequent quantitative PCR evaluation of silencing. In vit...

Construction of siRNA/miRNA expression vectors based on a one-step PCR process

BACKGROUND RNA interference (RNAi) has become a powerful means for silencing target gene expression in mammalian cells and is envisioned to be useful in therapeutic approaches to human disease. In recent years, high-throughput, genome-wide screening of siRNA/miRNA libraries has emerged as a desirable approach. Current methods for constructing siRNA/miRNA expression vectors require the synthesis...