Hi everyone,
I performed an assay following a protocol already established in the lab for a single homemade siRNA: 6 µL Dharmafect 1 mixed with siRNA in 200 µL serum free / antibiotic free medium prior to delivery on HeLa cells in 6 cm dish for 48h with final 100 nM of siRNA.
... now I've applied it to several smartpool on-TARGETplus freshly ordered from Dharmacon. And although I expected a strong silencing (100 nM...)... it's not working for everyone: gene 1 approx 70% of silencing, gene 2 approx 40% of silencing, gene 3... no silencing at all. Haven't looked at the protein expression yet.
Knowing that I've performed those in over 2-4 experiments so far. Always doing 2 different siRNA in parallel, so my constant silencing of gene 1 by 70% makes me confident about the delivery? I've used 2 different stock of siRNA, checked concentration, I've validated partial silencing on the protein level in previous experiment (different protocol)...
... am I missing something? is 100nM too much sometimes and counter-effective?
Any help will be highly welcomed,
Cheers,
Hello Benoit,
I made the experience in my siRNA transfections that if your sequences are not inducing a high silencing efficacy even the pooling of several sequences with average knock-down efficacy will not improve the outcome.
In addition it can depend on the gene itselfe. When you are knocking down an essential gene for your cells and it is highly regulated, transcription factors can strongly enhance the gene transcription and you end after 48 hours with 70% or so. In this case you can try to reduce the incubation time to 24-30 hours and have a look how it is initially working.
The siRNA amount is not that important for an effective siRNA. I made titration experiments with my siRNAs and ended with the same result as mentioned above. If the siRNA is moderately effective only 10 or 20 fold amounts will bring a statistical benefit but the knock-down effect will at best double. When you have a siRNA redcuing the gene expression around 30% as you have written above you can increase it at best to 50-60% when you increase the concentration 10 fold. The siRNA amount is more important than the concentration due to that the complexes precipate in around 6 hours.
Keep also an eye on the scrambled control siRNA you are transfecting. In the case you are using 100 nM final siRNA concentration this is quite enough I guess. What I have noticed is that the packing of the siRNA into the lipoplexes needs a certain concentartion of transfection reagent, siRNA amount in a certain volume to get working complexes for transfection. Maybe 200 µl for a 6 well is to less volume for a good transfection in such a big well. I used 100 µl final transfection mix with 40 nM as highest concentration in a 24 well and it worked very good. If you do RT-qPCR this well size is quite enought for 5 µg total RNA. I seeded 75,000 HeLa cells for 48 h and 125,000 for 24 h with a reverse transfection, first transfection mix into the well and then single cell suspenion added.
Are you making forward or reverse transfection? Reverse transfection will work a bit better. Have you made a Mycoplasama test? If Mycoplasma are present 90% of the effect turns ineffective. See:
https://www.researchgate.net/post/Has_anyone_performed_a_knockdown_via_siRNA_in_primary_keratinocytes#view=56e1e93d615e279bdf36b281
You should also take a look how the cells look like after the transfection in a time course. I knocked out an essential gene and HeLa started to die after 8 h and at the end I received a silencing of 50% due to that only cells who have survived supplied a huge proportion of the extracted RNA and the rest of the cells was degraded to apoptotic bodies. These cells I assume experienced a highly efficient knock-down responsible for thier fast death and no amplifyable RNA remained accounting for the moderate knock-down efficacy.
Hopefully my experiences can help you. Keep in mind that the cell starts to counteract a knock-down when an essential gene is hit and I think that is a likely explanation for your moderate knock-down efficacy. I have also siRNAs reducing only 40% of gene expression but cause a 60% reduced proliferation rate in HeLa because the gene is essential for them. In turn I have siRNAs causing a 98% reduced gene expresssion but the HeLa cells are totally unaffected in their growth capabilities because the gene is dispenable and even not tried to be reexpressed to normal levels.
Regards
Hello Benoit,
I made the experience in my siRNA transfections that if your sequences are not inducing a high silencing efficacy even the pooling of several sequences with average knock-down efficacy will not improve the outcome.
In addition it can depend on the gene itselfe. When you are knocking down an essential gene for your cells and it is highly regulated, transcription factors can strongly enhance the gene transcription and you end after 48 hours with 70% or so. In this case you can try to reduce the incubation time to 24-30 hours and have a look how it is initially working.
The siRNA amount is not that important for an effective siRNA. I made titration experiments with my siRNAs and ended with the same result as mentioned above. If the siRNA is moderately effective only 10 or 20 fold amounts will bring a statistical benefit but the knock-down effect will at best double. When you have a siRNA redcuing the gene expression around 30% as you have written above you can increase it at best to 50-60% when you increase the concentration 10 fold. The siRNA amount is more important than the concentration due to that the complexes precipate in around 6 hours.
Keep also an eye on the scrambled control siRNA you are transfecting. In the case you are using 100 nM final siRNA concentration this is quite enough I guess. What I have noticed is that the packing of the siRNA into the lipoplexes needs a certain concentartion of transfection reagent, siRNA amount in a certain volume to get working complexes for transfection. Maybe 200 µl for a 6 well is to less volume for a good transfection in such a big well. I used 100 µl final transfection mix with 40 nM as highest concentration in a 24 well and it worked very good. If you do RT-qPCR this well size is quite enought for 5 µg total RNA. I seeded 75,000 HeLa cells for 48 h and 125,000 for 24 h with a reverse transfection, first transfection mix into the well and then single cell suspenion added.
Are you making forward or reverse transfection? Reverse transfection will work a bit better. Have you made a Mycoplasama test? If Mycoplasma are present 90% of the effect turns ineffective. See:
https://www.researchgate.net/post/Has_anyone_performed_a_knockdown_via_siRNA_in_primary_keratinocytes#view=56e1e93d615e279bdf36b281
You should also take a look how the cells look like after the transfection in a time course. I knocked out an essential gene and HeLa started to die after 8 h and at the end I received a silencing of 50% due to that only cells who have survived supplied a huge proportion of the extracted RNA and the rest of the cells was degraded to apoptotic bodies. These cells I assume experienced a highly efficient knock-down responsible for thier fast death and no amplifyable RNA remained accounting for the moderate knock-down efficacy.
Hopefully my experiences can help you. Keep in mind that the cell starts to counteract a knock-down when an essential gene is hit and I think that is a likely explanation for your moderate knock-down efficacy. I have also siRNAs reducing only 40% of gene expression but cause a 60% reduced proliferation rate in HeLa because the gene is essential for them. In turn I have siRNAs causing a 98% reduced gene expresssion but the HeLa cells are totally unaffected in their growth capabilities because the gene is dispenable and even not tried to be reexpressed to normal levels.
Regards
Hi Volken Baumann, your explanation is really helpful. Do you perhaps have the publications related to this? Thank you
Here is the siPool Publication if you are interested in.
http://www.ncbi.nlm.nih.gov/pubmed/24875475
Thanks that my answer is a good helpful one :-)
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