I intend to make siRNA for my protein. I intend to send for the company to synthesis 3 difference region in the gene. Each region will be around 21 nucleotides. Then I will combine 3 single fragment of DNA and transfect into the cell.
After receiving the oligonucleotides, will I suspend into TE buffers as I did with primer or do I have to suspend in special buffer for siRNA? IF I have to suspend in special buffer for siRNA, where can I buy it? Or how can I make the buffer for siRNA?
Making the control siRNA, do you have any preferences sequences so that I don't have to make new control siRNA? In case of making control siRNA, could you give me any advice?
Many thanks
For what organism are you developing this? Is it really necessary to design your own sequences? I mean there are widely available pools of some 3-5 siRNA per gene. non-targeting siRNAs are usually against GFP/EGFP or luciferase, but also available.
I intend to knock down GEF1 in mouse (NIH3T3) and in human cell line (Hela)
After receiving the oligo nucleotides, will I suspend into TE buffes as I did with primer or I have to suspend in special buffer for siRNA?
If it's purified oligo (w/out salts) reconstitute in siRNA buffer, basic recipe is attached but I'd recommend you to order some just to be sure the solution is DNAse&RNAse free.
If the oligo lyophylisate already contains salts, reconstitute in DNAse&RNAse free water. This is up to manufacturer, check it. Addition of TE into 'salted' lyophylisate may slow down the dissolution and may lead to oligo precipitation.
http://dharmacon.gelifesciences.com/uploadedFiles/Resources/5x-sirna-buffer-protocol.pdf
Hello Nguyen,
I would recommend to take a look on this online tool for designing your siRNAs
http://dharmacon.gelifesciences.com/design-center/?redirect=true
I am also working with siRNAs but we can synthesize them in our institute so I use the online tool to find the sequences.
You can load the sequence of your gene or the accession number (I have not found a mouse gene with the annotation GEF1) and look for suitable siRNA sequences. Ordering is also possible or you copy the sequence and order it elsewhere.
The buffer to solubilize your siRNA when you have ordered can depend on the transfection reagent you want to use, but form my experiance it should not be very important. In all cases it should be nuclease free; I used nuclease free water from Sigma-Aldrich (W4502) or Thermo Scientific (R0582) for the stock solutions with 100 µM concentration and 1x DPBS (GIBCO, without CaCl2 and MgCl2 ,500 ml 14190-144 [cell culture media are also free of any enzymatic activity]) for the working solutions (10 or 1 µM) and it was stable for many freeze-thaw cycles > 30 (stock & working solutions).
The transfection reagent we are using is Lipofectamine 2000 Reagent and Lipofectamine RNAiMAX, when you want to transfect also plasmid vectors, Lipofectamine 2000 would be the better choice. siRNA and plasmids are tranfected very efficiently and the siRNA effect is stable for 3 days. That is the time span we are using for our proliferation experiments.
Keep in mind: When you use DNA sequences instead it will not work over the RNA inducing silencing complex!!!
Solubilize only in nuclease free liquids and autoclaved tubes (better, bought sterile from a company) and order RNA sequences!!!!
Good luck with you knock down!
PS: Pooling of sequences is not always benefitial! I have tried to pool two moderate effective (each, around 40% knock-down effect) siRNA sequences for one gene to test if the knock-down will be better compared to the single transfections. But it just increased to about 50% knock-down with both siRNA sequences. When you want to order 3 differnt sequences, you should test it in single transfecitons if one sequence is really potent (knock-down effect is >70%, better 80-90%). Then you should use this single sequence, using the others too, will only increase artificial side effect and maybe be disadvantagous for your experiment!
Volker is right, pooling unvalidated siRNAs may play a little with miRNAs and affect the efficiency of knock-down, please, check also another RNAi related questions for that, it was already discussed few times.
When I order the oligo synthesis: I only order the sense?
For example: I have 3 short sequences at 3 regions in one gene. Do I have to order the antisense together. After that I will combine sense and antisense together? Or only sense sequence is enough like the sample below?
1.AAA UUA AGA ACA CGU CUG UCU GCGG
2.UUU GAC AGC ACC GAU CUC UUU CGC C
3.UUG GUC GUG UAA CUG AUG AGC AGG C
You need both sense and antisense for 2 reasons
1. the RNAi machinery loads dsRNA and may not be able to recognize free ssRNA.
2. ssRNA is very unstable and may not survive long enough to make it into the RISC.
When you buy siRNA from companies like Dharmacon you would already get them as a duplex. You may also may want to consider using shRNA instead of synthetic RNA.
When you order you have to order both strands, this is already the issue when you order siRNAs you get them duplexed in different amounts 5, 10, 20 nM.
You have 19 bp in your siRNA sense strand fitting to the mRNA sequence of your desired gene, and two UU or TT (DNA translated) which are the overhang, in sum 21 bp.
Dharmacon also add two UU RNA nucleotides to further stabilze the siRNA duplex against degradation, this is also not affecting the RISC complex or knock-down efficiency.
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