You're going to have to give us more details. What IVT kit do you use, how do you re-aneal your RNA, etc. ?

Hello, i did various ways. I have used Ambion Megascript for IVT as well as purification, Ampliscribe for IVT as well as purification and also Ambion for IVT and 5M ammonium acetate for purification. Finally the latter worked after pooling my PCR products during PCR purification step. I got about 7.9 microgram/microlitre and injected the same. For IVT- 32oC for 6 hrs, 85oC for 30 min and 25oC for 10 min. The question is that iam not able to get any significant knockdown of my gene at such a concentration as well. Please suggest.

THANKS

You still have not answered the question how you anneal your RNA after the IVT?

Hello, After IVT, i incubate the reaction at 75oC for 5-10 min, then cooling to room temperature for annealing RNA. Though, it has been known that it may not be necessary as my gene is less than 800 nt and the two strands were synthesized from single transcription templates.

For annealing you need 65 for 30 min or 95-100 for 5 min to denature , then you need slow cooling to RT. that means either programing a PCR machine, or having an enclosed environment, left to cool down slowly. The salt composition of the solution can also influence annealing efficiency. Do you check your RNA yield before annealing? You should. That will tell you whether your problem is with the IVT reaction or with the annealing. If the problem is not with the annealing but your yield is low you can try to do/check several things:

1. Make sure you got the right polymerase e.g. T7, T3, etc.

2. Make sure you ste up the reactions at RT!!!!! The spermidine in the Txn buffer will precipitate at lower temperatures, that also means you have to make sure it is in solution ( you don't see any white flaky stuff) before you add it to reaction. Also don't add plasmid and 10X Txn buffer, before adding water.

3. Try to Increase amount of DNA and/or decrease incubation time, maybe at 6 h you have some of your RNA degraded, do you have an Rnase inhibitor in your reaction, do you use Dnse, RNase free water? If you suspect RNA degradation, run your completed reaction of a gel, it will tell you whether you have degradation.

4. Is you IVT kit a high-efficiency kit? That shouldn't matter much, but it could. We obtain similar results with high-efficiency kits (promega) and just regular T7 pol, but if you already have some issues, maybe you want to switch to a high-efficiency kit.

Finally, as long as you do not have RNA degradation, if all else fails, just combine several reactions together to obtain sufficient amount of dsRNA.

Hello, i truly appreciate your troubleshooting guide. But i would like to continue with the discussion-

1- I will surely try with your annealing suggestions, slow cooling is always done in PCR (25oC for 30 min.)

2- I will check RNA yield just before annealing, i used to do after annealing and run the gel to check the yield.

3- Polymerase is with Ambion Megascript Kit (T7), reaction started at RT

4- I will try to decrease the incubation time, don't have an RNAse inhibitor in the reaction, but Nuclease-free water.

5- I have not tried with Promega, just using Ambion/Ampliscribe.

I did mix several reactions together to get sufficient amount of dsRNA, say i could mix three seperate IVT reactions and get 8 microgram/microlitre of purified dsRNA. But for my gene, it is also not working and in some cases it may be 100 micrograms. So i have to increase the yield drastically.

Also do PCR product (purified) work fine as templates for IVT reaction. or can you suggest a procedure for linearizing the template, i have tried with double digests.

Please suggest.

Slow cooling means a slow rate of temperature decrease, that is to get from 65 to 25 very slowly.

What is the target of your dsRNA? Do you add your dsRNA to cells in culture? I don't understand what is not working, you need to be more clear. Please answer the following:

1. What is your dsRNA yield per microgram of DNA template

2. Where are you adding your dsRNA?

3. How many micrograms of dsRNA are you adding to deplete your protein?

4. How long does your dsRNA treatment last?

5. How long is your dsRNA? How big is your gene?

Hello, Let me answer your more pertinent questions-

1-dsDNA concentration taken into IVT reaction- 0.380 microgram/microlitre, dsRNA yield after pooling three separate IVT reactions- 7.92 microgram/microlitre.

That's the best i have achieved.

2- dsRNA is injected to larvae of beetle once (1 microlitre injection).

3- Currently iam using about 8 micrograms of dsRNA to deplete the protein.

4- I usually observe the knockdown by RT-PCR analysis with dsRNA primer till 7-8 days after injection.

5- dsRNA will be about 350 bp (including the T7 promoter sequence). The gene is an important cellular signaling molecule in the Toll pathway of insect and is about 1172 bp.

Please find the results-4 days post injection of dsRNA. LANE 1-4: L27A (housekeeping); LANE 6-9: target gene (6,7-buffer injected; 8,9- dsRNA injected)

Hope it solves.

Please suggest.

Dear Bharat, as far as I understood you're not having problems with the yeld of dsRNA production, but with the knockdown efficiency, is that correct? 8 ug of dsRNA should be more than enough for silencing a gene.

In order to be more specific, I'd need you to answer the questions below.

Which set of primers do you use for RT-PCR to check RNAi efficiency?

The same dsRNA primers or a different pair of primers?

In which position do they anneal in your gene, compared to the position of the PCR fragment (e.g. next to our PCR fragment, within it, etc.)?

Best.

Thanks for replying. Please find my answers appended below-

1- I have been using the same dsRNA primers designed with the T7 promoter region used for PCR amplification to check RNAi efficiency. The product size for checking the RNAI efficiency will be the same. The results were inconsistent with 8 microgram of dsRNA. Lately, we have ordered a monitoring primer to check for RNAi efficiency that overlaps the region of knockdown but not completely of the same size.

2- They anneal to the PCR fragment knocked down, but the monitoring primer ordered lately, only anneal a part of the silenced gene.

Please suggest further.

Hi, thanks for a lively discussion. I got a knockdown of more than 75% for two genes- one a membrane protein and the second a critical component of intracellular signaling pathway. The first one is a result of template from plasmid cut with BST XI and the second one by PCR amplification of direct plasmid. Both works but with a primer designed from a seperate overlap region as opposed to the primer used to make dsRNA.

Good luck