RNAi in a sense/antisense transcript system - (Dec/05/2005 )
Hi
I want to study the regulation of a sense/antisense transcript system. My plan is to knock down the sense or the antisense transcript via siRNAs and see the other transcript go up (measured by RT-PCR). So I have some questions about that.
- Unfortunately I can place my siRNAs only in a region where both transcripts are present. What about strand specifcity of the siRNA? I have to be sure, that the siRNA only knocks down the sense or the antisense transcript but not the complementary transcript.
- I prefer to use synthesized RNA oligos because I think it´s the quicker way (compared to clone own siRNA Vectors) to do the experiments. How much of RNA oligo I will need per transfection, let´s say with 10 cm cell dishes? I read in a Quiagen manual that you have to use nearly all of your oligo for transfecting one well of a 20 well plate. I am a little bit worried about that.
- Would you suggest me to use another method than ordering oligos? What about siRNA Vectors? It would take some time to clone them, but on the other hand I woudn´t have any limitations regarding the amount of siRNA and transfection of Plasmid DNA to my cells (U87) is already established.
- What about controls and number of siRNAs per target transcript? How many siRNAs should I test to be sure to get a significant knockdown and of course I want to minimize costs and/or cloning effort? Do I need only a single random siRNA as negative control or one for every target siRNA?
Many thanks for your help.
Morrison
hi
that is an interesting investigation.
as principle of siRNA, the duplex leads to a triple helix and only one strand of the uplex serves for the specificity. so the underlying question your follow is : if target is antisense, does the opposite strand serves as the probe to target RNA?
Hi Morrison,
- Unfortunately I can place my siRNAs only in a region where both transcripts are present. What about strand specifcity of the siRNA? I have to be sure, that the siRNA only knocks down the sense or the antisense transcript but not the complementary transcript.
A: Yes, there is such concern if you want to only silence one transcription. Actually, both strands of siRNA duplex have equal chance of entering the RISC complex. There are several papers reporting ways of letting one particular strand be used as the guide strand. Generally, if the 3' end of the duplex is less themodynamically stable (with less GC), the antisense strand will have greater chance entering RISC (Khvorova et al Cell 2003, Schwarz et al Cell 2003). Add a 5' phosphate group to the antisense strand can also help RISC identify it as guide strand. Lastly, design a mismatch at the 3' most basepair of the duplex will also help RISC recognize the antisense strand as the guide strand.
- I prefer to use synthesized RNA oligos because I think it´s the quicker way (compared to clone own siRNA Vectors) to do the experiments. How much of RNA oligo I will need per transfection, let´s say with 10 cm cell dishes? I read in a Quiagen manual that you have to use nearly all of your oligo for transfecting one well of a 20 well plate. I am a little bit worried about that.
A: I agree with you. Let me give you some general idea. If I order 20 nM siRNA, I can make 1 ml of 20 uM stock siRNA solution from it. If I do transfection in 6 well plate at the concentration of 50 nM, I only need 6.25 ul of 20 uM siRNA for each well. For a 100-mm dish, I need about 30 ul. So you can calculate how many transfections you can do with just one order which costs about $300.
- Would you suggest me to use another method than ordering oligos? What about siRNA Vectors? It would take some time to clone them, but on the other hand I woudn´t have any limitations regarding the amount of siRNA and transfection of Plasmid DNA to my cells (U87) is already established.
A: If you want to use shRNA, check sigma, they are selling cloned shRNA libraries.
- What about controls and number of siRNAs per target transcript? How many siRNAs should I test to be sure to get a significant knockdown and of course I want to minimize costs and/or cloning effort? Do I need only a single random siRNA as negative control or one for every target siRNA?
A: Make sure your control siRNA doesn't have homology with any sequences and one control should be enough and can be used for different gene. Test at least 2 siRNAs for your gene.
Good luck.
Eric
Thank you for your help.
I think I will order oligos and try to design them so that they would be strand specific. I hope it works.
Morrison