VECTOR-BASED siRNA FAQ1. What is a vector-based siRNA?Vector-based siRNA is a novel technology that generates siRNA effects using DNA vector and a small hairpin insert. In DNA vector-based siRNA technology, a small DNA insert (about 70 bp) encoding a short hairpin RNA targeting the gene of interest is cloned into a commercially available siRNA vector. The insert-containing vector can be transfected into the cell, and it expresses the short hairpin RNA. The hairpin RNA is rapidly processed by the cellular machinery into 19-22 nt double stranded RNA (siRNA).2. What is the difference between vector-based siRNA and siRNA cassette?Vector-based siRNA has a selection marker in the vector, and it allows you to establish stable cell lines. siRNA cassette is a PCR product, and it is used for transient experiments and cannot be used for establishing stable cell line.3. Why does the hairpin insert sequence need to start with an 'A' or 'G'?U6 and H1 are RNA Polymerase Type III promoters. RNA Polymerase type III promoters prefer a purine (A or G) for transcription. Therefore, if the hairpin insert sequence does not start with an 'A' or 'G', a 'G' will be added in the front of the hairpin insert sequence.4. Have the target sequence been filtered against other homologous sequences in the genome?When you input the sequence in genscript siRNA design center, our computer is calculating in the background to filter out all the homologous sequence in the genome so that the target sequence is specific. More than that, the ranking also reflects the specificity of a siRNA target site.5. What are the promoters of genscript vectors?Genscript vectors use two kind of promoters: human U6 and human H1.6. What are the difference between U6 and H1 promoters?In our internal testing, we have not found significant difference between U6 and H1 promoters in CHO, HEK293, and Hela cells. However, it is recommended that you test both promoters in your particular cell line.7. Do human U6 and H1 work in rat, mouse or other mammalian cells?Yes. There is no species difference for human U6 and H1 promoters. These promoters work very well in rat, mouse or other mammalian cells.8. Can genscript vectors be used in Chicken, Plant, and Drosophila?Genscript vectors have not been tested in Chicken, Plant or Drosophila. We are not sure whether they work or not.9. Will you guarantee that the recommended siRNA constructs work?By using genscript siRNA design center and custom siRNA, there is 95% chance that you will find a potent siRNA target (70% inhibition) if you test 3 siRNA constructs for a gene. The most common problem for siRNA failure is the transfection efficiency. Genscript has developed GFP marker vector to address that issue. However, genscript does not guarantee that the recommended siRNA constructs will work because of uncertainty in the transfection experiments.10. How many constructs do you recommend to order for a gene?We recommend that at least 3 siRNA constructs should be tested for each gene.11. How do I monitor the transfection efficiency?You can monitor the transfection efficiency indirectly by co-transfecting a GFP plasmid. Alternatively, you can use genscript cGFP marker siRNA vector (pRNAT series) to directly monitor the transfection efficiency.12. What is cGFP?cGFP is a coral GFP developed by genscript Corporation. Its fluorescence intensity is similar to other commercial GFP proteins such as EGFP, and cycleGFP. Its excitation wavelength is 487 nm, and its emission wavelength is 507 nm.13. Can genscript make vector-based siRNA constructs using the vectors from other companies?Yes. You have to provide the vectors.14. What kind of negative control do you recommend?For negative controls, you can use our siRNA against firefly luciferase since firefly luciferase gene may not exist in the organism you worked. Alternatively, genscript has developed a sequence scrambler to make a specific negative control for your siRNA.15. How long is the delivery time for vector-based siRNA?The delivery time is about 3 weeks.16. What antibiotic should I use for neomycin selection marker?G418 is the recommended antibiotic for neomycin.17. Before transfection, can I linearize the plasmid? Which enzyme should I use to linearize the vector?Yes, you can use BglII or MulI to linearize the vector before transfection, which may help in integrating into chromosomes.18. Why is your default design is antisense-loop-sense, not the reverted one?The anisense-loop-sense design shows more potency according to our experiments. However, if you prefer sense sequence + loop + anti-sense sequence design, please feel free to let us know and we have no problem to do it for you.19. What's the advantage of using adenoviral transfection?The virus-mediated RNAi may circumvent some of the problems associated with cells that are generally refractory to RNAi, such as nontransformed primary cells, e.g. fibroblasts and dendritic cells.20. Why do you set the default length to 21-mer instead of 19-mer?21-mer is better based on the following reference: Harborth J, Elbashir SM, Vandenburgh K, Manninga H, Scaringe SA, Weber K, Tuschl T. (2003) Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev. 2003 Apr;13(2):83-105.21. Which inducer is better? Tetracycline or doxycycline?Doxycycline is better, because it is more stable in the medium. Tetracycline can be degraded easily.22. Why do you add one more C after BamHI site in the siRNA insert?The transcription of pol III promoter starts at position 8. That extra C is to make sure that the siRNA insert gets transcribed correctly.23. Why is my lentiviral virus titer from pRNA-U6.1/lenti or pRNATin-H1.2/lenti low?It is normal since the transfection efficiency and the copy number transfected is all different from cell to cell. Just make sure optimal ratio of target plasmid and packaging plasmids. The lentiviral titer is lower than other viral system. Invitrogen people suggest that transfection of 293FT packaging cell in suspension may improve the titer. I attached the protocol. And also you may transduce several times with lower titer stock every other day in order to accumulate cGFP positive cells. Make sure cells are not completely confluent in order to maintain active growing condition and during this series of transductions you may split cells.订购信息
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