Frequently Asked Questions: Vector-Based siRNA

1. What is vector-based siRNA?
2. What are the advantages of vector-based siRNA?
3. What is the difference between vector-based siRNA and siRNA cassettes?
4. What are the criteria for siRNA target site design?
5. Why does the hairpin insert sequence need to start with an adenine or guanine?
6. Has the target sequence been filtered against other homologous sequences in the genome?
7. What promoters do GenScript vectors use?
8. What are the differences between U6 and H1 promoters?
9. Do human U6 and H1 work in rat, mouse, or other mammalian cells?
10. Can GenScript vectors be used in chicken, Drosophila, or plant cells?
11. Does GenScript provide adenoviral and retroviral siRNA vectors?
12. Does GenScript provide synthetic siRNA oligos?
13. Does GenScript guarantee that the recommended siRNA constructs will work?
14. How many constructs should be made per order?
15. What is the siRNA construct transfection protocol?
16. Which transfection reagents work best with siRNA vectors?
17. How can transfection be confirmed?
18. How can transfection efficiency be monitored?
19. What is cGFP?
20. Can GenScript make vector-based siRNA constructs using vectors from other companies?
21. Does vector-based siRNA work with in vivo studies?
22. What kind of negative control is recommended?
23. What is the delivery time for vector-based siRNA?
24. What are the fees for shipping outside the US?
25. How can I place an order?
26. What kind of payments do you accept?
27. Can I use oligofectamine to perform the transfection?
28. Where can I order antibiotics for the zeomycin selection marker?
29. Which antibiotic should I use with the neomycin selection marker?
30. Can I linearize the plasmid before transfection? Which enzyme should I use?
31. Why is the default design antisense-loop-sense and not sense-loop-antisense?
32. What are the advantages of adenoviral transfection?
33. Why is the default length 21-mer and not 19-mer?
34. Which inducer is better, tetracycline or doxycycline?
35. Can I also order the bacteria containing the plasmid for future plasmid isolation?
36. Why is there an extra cytosine after the BamHI site in the siRNA insert?
37. Why is my lentiviral virus titer from pRNA-U6.1/lenti or pRNATin-H1.2/lenti low?

1. What is a vector-based siRNA?
   Vector-based siRNA is a novel and resilient means of harnessing the effects of siRNA and RNAi without costly and cumbersome traditional siRNA synthesis. In DNA vector-based siRNA technology, a small DNA insert encoding a short hairpin RNA sequence that targets the gene of interest is cloned into a commercially available siRNA vector. The insert-containing vector can be transfected into the cell, where it expresses the short hairpin RNA. The hairpin RNA is rapidly processed by the cellular machinery into a 19- to 22-nucleotide double-stranded RNA (siRNA).
2. What are the advantages of vector-based siRNA?
   Vector-based siRNA is more stable and easier to handle than traditional siRNA. Since it is absorbed into the host cell's DNA sequence, it can be used to establish stable cell lines and knockout mouse lines. Please click here to learn more.
3. What is the difference between vector-based siRNA and siRNA cassettes?
   Vector-based siRNA allows for the establishment of stable cell lines and each vector comes with a selection marker. siRNA cassettes are PCR products. They cannot establish stable cell lines but may be well suited to more transient experiments.
4. What are the criteria for siRNA target site design?
   siRNA target design takes ten different factors into account. These include the thermodynamic properties of the construct in question, its length, secondary structure, and intended host, and the GC content of the target. Please click here for a more detailed description.
5. Why does the hairpin insert sequence need to start with an adenine or guanine?
   U6 and H1 are RNA Polymerase Type III promoters, which prefer a purine (adenine or guanine) for transcription. If the hairpin insert sequence does not start with an A or G, a G is added to the front of the hairpin insert sequence.
6. Has the target sequence been filtered against other homologous sequences in the genome?
   Yes. The GenScript siRNA Design Center computer filters out all homologous sequences so that the construct responds to a specific target site. In addition, the ranking displayed on the screen also reflects the degree of specificity.
7. What promoters do GenScript vectors use?
   GenScript vectors use human U6 and human H1 promoters.
8. What are the differences between U6 and H1 promoters?
   In our internal testing, we have not found any significant difference between U6 and H1 promoters in CHO, HEK293, and Hela cells. However, we recommend testing both promoters in the particular cell line to be used.
9. Do human U6 and H1 work in rat, mouse, or other mammalian cells?
   Yes. These promoters work very well in rat, mouse, and other mammalian cells.
10. Can GenScript vectors be used in chicken, Drosophila, or plant cells?
    GenScript vectors have not been tested in chicken, Drosophila, or plant cells.
11. Does GenScript provide adenoviral and retroviral siRNA vectors?
    Yes. GenScript provides a wide range of vectors, including plasmidic, inducible, and viral vectors.
12. Does GenScript provide synthetic siRNA oligos?
    No. GenScript does not provide synthetic siRNA oligos. However, GenScript vector-based siRNA can meet all the same needs as synthetic oligos and then some.
13. Does GenScript guarantee that the recommended siRNA constructs will work?
    About 95% of the custom siRNA constructs made by GenScript and designed at the GenScript siRNA Design Center show potent target inhibition, 70% or better, when tested in groups of three. The most common cause of siRNA failure is low transfection efficiency. To deal with this problem, GenScript has developed GFP-marked vectors to track and confirm transfection efficiency. However, because of the uncertainly involved in transfection experiments, GenScript does not guarantee that all its siRNA constructs will work.
14. How many constructs should be made per order?
    We recommend that at least three siRNA constructs be tested for each gene.
15. What is the siRNA construct transfection protocol?
    Download the protocol here.
16. Which transfection reagent works best with siRNA vectors?
    GenScript recommends using Lipofectamine^TM 2000, from Invitrogen, with siRNA vectors.
17. How can transfection be confirmed?
    GenScript offers positive controls that help optimize transfection procedures. Please use this link to learn more.
18. How can transfection efficiency be monitored?
    Transfection efficiency can be monitored indirectly by co-transfecting a GFP plasmid. GenScript also offers siRNA vectors with cGFP markers built in. These vectors, the pRNAT series, allow for direct monitoring of transfection efficiency.
19. What is cGFP?
    cGFP is a coral green fluorescent protein developed by GenScript Corporation. The intensity of its fluorescence is similar to that of other commercial GFP proteins such as EGFP (Enhanced Green Fluorescence Protein). Its excitation wavelength is 487 nm, and its emission wavelength is 507 nm.
20. Can GenScript make vector-based siRNA constructs using vectors from other companies?
    Yes. The client must either provide the vector or pay a reasonable fee.
21. Does vector-based siRNA work with in vivo studies?
    Yes. Vector-based siRNA has been used to make transgenic mice, sometimes by direct injection.
22. What kind of negative control is recommended?
    For negative controls, we recommend using our siRNA against firefly luciferase, assuming that the firefly luciferase gene does not exist in the organism under study. Alternatively, we have developed a sequence scrambler that creates negative control constructs specific to any siRNA construct. Please use this link to learn more.
23. What is the delivery time for vector-based siRNA?
    Turnaround time is generally three weeks.
24. What are the fees for shipping outside the US?
    The fee for shipping outside the US is generally $35 per shipment.
25. How can I place an order?
    GenScript needs both the desired siRNA sequence and payment information. We accept both PO (Purchase Order) numbers and credit cards for online orders. Alternatively, the customer can send us the sequence by email and fax us the PO. All clients are welcome to use the GenScript siRNA Design Center.
26. What kind of payments do you accept?
    GenScript accepts credit cards (Visa, MasterCard, American Express), checks, and wire transfers. For orders, the customer may send us a PO (Purchase Order). For certain orders, GenScript may require a deposit.
27. Can I use oligofectamine to perform the transfection?
    We have not tested oligofectamine with our vector-based siRNA.
28. Where can I order antibiotics for the zeomycin selection marker?
    Order Zeocin^TM Selection from Invitrogen (Cat # R25005).
29. Which antibiotic should I use with the neomycin selection marker?
    G418 is the recommended antibiotic for use with neomycin.
30. Can I linearize the plasmid before transfection? Which enzyme should I use?
    Yes. Use BglII or MulI to linearize the vector before transfection. This may help the construct integrate into the chromosomes.
31. Why is the default design antisense-loop-sense and not sense-loop-antisense?
    According to our experiments, the antisense-loop-sense design shows more potency. However, if the customer prefers sense-loop-antisense sequence design, it can be provided easily.
32. What are the advantages of adenoviral transfection?
    Virus-mediated RNAi may circumvent some of the problems associated with certain types of cells, such as nontransformed primary cells, like fibroblasts and dendritic cells.
33. Why is the default length 21-mer and not 19-mer?
    We have selected 21-mer as our default size based on the following references: Harborth J, Elbashir SM, Vandenburgh K, Manninga H, Scaringe SA, Weber K, Tuschl T. 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.
34. Which inducer is better, tetracycline or doxycycline?
    Doxycycline is more stable in the medium. Tetracycline can be degraded easily.
35. Can I also order the bacteria containing the plasmid for future plasmid isolation?
    Yes. Customers can purchase the bacteria for an additional $100 fee.
36. Why is there an extra cytosine after the BamHI site in the siRNA insert?
    The transcription of Pol III promoter starts at position 8. That extra cytosine ensures that the siRNA insert is transcribed correctly.
37. Why is my lentiviral virus titer from pRNA-U6.1/lenti or pRNATin-H1.2/lenti low?
    The transfection efficiency and the copy number may differ from cell to cell. Make sure to use an optimal ratio of target plasmids to packaging plasmids. The lentiviral titer is lower here than in other viral system. Invitrogen suggests that transfection of 293FT packaging cells in suspension may improve the titer. Also, the user may transduce several times with a lower titer stock every other day in order to accumulate cGFP-positive cells. Make sure that the cells are not completely confluent to maintain active growing conditions. During this series of transductions, the cells may be split.