Product Cart
| Cat#: | SI606B-1-GVO-SBI |
| Quantity: | 2 x 10^7 ifu, 2-3 Transductions |
| Price: | 6874 € |
| Supplier: | SBI |
| Please note: | Genetically modified organism (GVO), see General Terms. |
- 200,000 siRNA sequences targeting 47,400 human mRNA transcripts
- Selectable puromycin resistance allows positive identification and selection of transduced cells
- Pre-packaged, ready-to-use format—transduce your cells the day you receive the library
- High-throughput identification of functional siRNAs by Affymetrix GeneChip® hybridization
The GeneNet Human 50K siRNA Library contains 200,000 siRNA templates targeted to 47,400 human transcripts listed in the NCBI RefSeq database. For most of the target genes, four different siRNA sequences were designed using SBI´s proprietary algorithm. The library is cloned into HIV-based pSIH1-H1-Puro shRNA Expression Vector, which confer resistance to the antibiotic puromycin. This single-promoter siRNA vector expresses cloned short-hairpin RNA (shRNA) from a single H1 RNA polymerase III promoter.
The pre-packaged VSV-G pseudotyped viral stock can be used to transduce your cells of choice immediately upon receipt, or be stored at -70oC for several months.
After transducing the library into your cells of interest, you select those cells that display the desired phenotype or response using whichever technique is appropriate for your system.
Following selection, the lentiviral inserts—containing the siRNA templates—are easily recovered by PCR and identified using the GeneChip® Human Genome U133 Plus 2.0 Array (Cat.# 900470) manufactured by Affymetrix (Santa Clara, CA). The siRNA template sequences were designed to hybridize to oligonucleotide probes on this array to facilitate identification. Although more tedious, you can also use sequencing to identify the particular siRNA sequences in selected cells.
To confirm representation, siRNA templates for the library were packaged in viral particles, amplified, labeled, and hybridized to the Affymetrix Human Genome U133 Plus 2.0 Array. Similarly, siRNA templates were amplified from the genomic DNA extracted from target cells transduced with the library. These templates were also labeled and hybridized to the Human Genome U133 Plus 2.0 Array. Overall, in both the viral- and cell-derived samples, more than 55% of the gene-specific siRNA template species used in construction of the library were easily detectable above background. In addition, the correlation of the relative amounts of different siRNA template species between both samples was very high, indicating that there is not appreciable loss or change in representation with transduction. These results indicate that the library delivers a representative number of siRNA sequences that target the majority of genes for which the library was originally designed. Also, the high correlation between the pre- and post-transduction samples shows that the pseudotyped viral particles transduce with similar efficiency, regardless of the siRNA template they harbor.
Product Components
Cat. # SI606B-1
500-2000 µl GeneNet™ Human 50K siRNA Library, pre-packaged in VSV-G particles (2x10e7 ifu)
50 µl cDNA Synthesis GNF/GNH Primer (10 µM)
50 µl Fwd GNF/GNH (Forward) PCR Primer (10 µM)
50 µl Rev GNF/GNH (Reverse) PCR Primer (10 µM)
200 µl NFwd-Bio (Nested Forward Biotinylated) PCR Primer (10 µM)
200 µl NRev GNF/GNH (Nested Reverse) PCR Primer (10 µM)
200 µl NRev GNF1/GNH1 (Nested Reverse 1) PCR Primer (10 µM)
200 µl NRev GNF2/GNH2 (Nested Reverse 2) PCR Primer (10 µM)
200 µl NRev GNF3/GNH3 (Nested Reverse 3) PCR Primer (10 µM)
20 µl Positive Control DNA (isolated from GeneNet Mouse 40K siRNA library) (100 pg/µl)
100-500 µl Packaged Positive Transduction Control (>2x105 ifu)
1 disc CD with gene/siRNA list, and data analysis program compatible with Affymetrix data file
References
Hattori, H. et al. (2007) RNAi screen identifies UBE2D3 as a mediator of all-trans retinoic acid-induced cell growth arrest in human acute promyelocytic NB4 cells. Blood 110: 640-650.
Hwang G.W. et al. (2007) siRNA-mediated inhibition of phosphatidylinositol glycan Class B (PIGB) confers resistance to methylmercury in HEK293 cells. J Toxicol Sci. 32: 581-583.
