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Antisense oligonucleotides versus siRNA Hi, I would like to knock down a gene in vivo in rat (intrathecal injection). What are differences between antisense oligonucleatides and siRNA?...
Stable siRNA transfection HI, I do a lot of siRNA transfection for expts like PI staining, protein extracts, western blot etc wherein I require a lot of cells. The main...
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Effect of p27Kip1 inhibition on proliferation of bovine corneal endothelial c... Related Articles

Effect of p27Kip1 inhibition on proliferation of bovine corneal endothelial cells by RNA interference.

J Huazhong Univ Sci Technolog Med Sci. 2008 Apr;28(2):211-5

Authors: Huang Y, Zhang M, Wang Y, Fan K, Zhang G, Zhou Y

Three plasmids (pGenesil-P1, pGenesil-P2, pGenesil-P3) with different p27Kip1-shRNA sequences were designed and synthesized. Their effects on the proliferation of bovine corneal endothelial cells (bCEC) were investigated. Plasmid expressing irrelevant shRNA with a random combination was used as negative control (pGenesil-HK). The recombination of four plamids was confirmed by restrictive enzyme digestion and sequence analysis. The expression of mRNA and protein of p27Kip1 was detected by RT-PCR and Western blotting after stable transfection. The expressions of p27Kip1 mRNA and p27Kip1 protein of pGenesil-P1 group, pGenesil-P2 group and pGenesil-P3 group were all lower than those in the pGenesil-HK group and the blank group (non-transfected group). pGenesil-P3 had the strongest inhibitory effect and was selected for the next steps. The proliferation rates of the pGenesil-P3 group, the pGenesil-HK group and the blank group were assessed by MTT. The influence of shRNA-p27Kip1 on bCEC cell cycle was detected by flow cytometry (FCM). Compared with the control groups, the proliferation rate of the pGenesil-P3 group was increased significantly, and the ratio of S-phase also increased. It is concluded that shRNA-p27Kip1 could down-regulate the expression of p27Kip1 effectively and increase the proliferation of bCEC. RNA interference (RNAi) may be an effective means to promote the proliferation of CEC.

PMID: 18481001 [PubMed - in process]

Integrating Experimental and Analytic Approaches to Improve Data Quality in G... Related Articles

Integrating Experimental and Analytic Approaches to Improve Data Quality in Genome-wide RNAi Screens.

J Biomol Screen. 2008 May 14;

Authors: Zhang XD, Espeseth AS, Johnson E, Chin J, Gates A, Mitnaul LJ, Marine SD, Tian J, Stec EM, Kunapuli P, Holder DJ, Heyse JF, Strulovici B, Ferrer M

RNA interference (RNAi) not only plays an important role in drug discovery but can also be developed directly into drugs. RNAi high-throughput screening (HTS) biotechnology allows us to conduct genome-wide RNAi research. A central challenge in genome-wide RNAi research is to integrate both experimental and computational approaches to obtain high quality RNAi HTS assays. Based on our daily practice in RNAi HTS experiments, we propose the implementation of 3 experimental and analytic processes to improve the quality of data from RNAi HTS biotechnology: (1) select effective biological controls; (2) adopt appropriate plate designs to display and/or adjust for systematic errors of measurement; and (3) use effective analytic metrics to assess data quality. The applications in 5 real RNAi HTS experiments demonstrate the effectiveness of integrating these processes to improve data quality. Due to the effectiveness in improving data quality in RNAi HTS experiments, the methods and guidelines contained in the 3 experimental and analytic processes are likely to have broad utility in genome-wide RNAi research. (Journal of Biomolecular Screening XXXX:xx-xx).

PMID: 18480473 [PubMed - as supplied by publisher]

The potato-specific apyrase is apoplastically localized and has influence on ... Related Articles

The potato-specific apyrase is apoplastically localized and has influence on gene expression, growth and development.

Plant Physiol. 2008 May 14;

Authors: Riewe D, Grosman L, Fernie AR, Wucke C, Geigenberger P

Apyrases hydrolyze nucleoside triphosphates and diphosphates and are found in all eukaryotes and a few prokaryotes. Although their enzymatic properties have been well characterized, relatively little is known regarding their subcellular localization and physiological function in plants. In the present study we used reverse genetic and biochemical approaches to investigate the role of potato-specific apyrase. Silencing of the apyrase gene family with RNAi-constructs under the control of the constitutive 35S-promoter led to a strong decrease in apyrase activity to below 10% of the wild-type level. This decreased activity led to phenotypic changes in the transgenic lines including a general retardation in growth, an increase in tuber number per plant and differences in tuber morphology. Silencing of apyrase under the control of a tuber-specific promoter led to similar changes in tuber morphology, however, there were no direct effects of apyrase inhibition on tuber metabolism. DNA micro-arrays revealed that decreased expression of apyrase leads to increased levels of transcripts coding for cell wall proteins involved in growth and genes involved in energy transfer and starch synthesis. To place these results in context, we determined the subcellular localization of the potato-specific apyrase. Using a combination of approaches we were able to demonstrate that this enzyme is localized to the apoplast. We describe the evidence which underlies both this fact and that potato-specific apyrase has a crucial role in regulating growth and development.

PMID: 18480378 [PubMed - as supplied by publisher]

AsiDesigner: exon-based siRNA design server considering alternative splicing. Related Articles

AsiDesigner: exon-based siRNA design server considering alternative splicing.

Nucleic Acids Res. 2008 May 14;

Authors: Park YK, Park SM, Choi YC, Lee D, Won M, Kim YJ

RNA interference (RNAi) with small interfering RNA (siRNA) has become a powerful tool in functional and medical genomic research through directed post-transcriptional gene silencing. In order to apply RNAi technique for eukaryotic organisms, where frequent alternative splicing results in diversification of mRNAs and finally of proteins, we need spliced mRNA isoform silencing to study the function of individual proteins. AsiDesigner is a web-based siRNA design software system, which provides siRNA design capability to account for alternative splicing for mRNA level gene silencing. It provides numerous novel functions including the designing of common siRNAs for the silencing of more than two mRNAs simultaneously, a scoring scheme to evaluate the performance of designed siRNAs by adopting currently known key design factors, a stepwise off-target searching with BLAST and FASTA algorithms and checking the folding secondary structure energy of siRNAs. To do this, we developed a novel algorithm to evaluate the common target region, where siRNAs can be designed to knockdown a specific mRNA isoform or more than two mRNA isoforms from a target gene simultaneously. The developed algorithm and the AsiDesigner were tested and validated as very effective throughout widely performed gene silencing experiments. It is expected that AsiDesigner will play an important role in functional genomics, drug discovery and other molecular biological research. AsiDesigner is freely accessible at http://sysbio.kribb.re.kr/AsiDesigner/.

PMID: 18480122 [PubMed - as supplied by publisher]