Hello Arnab,

An simple explanation is, if the gene/protein is very essential for the cell type you are using, then you see a decrease in the protein level after 72h but the RNA level is back at the normal level after 72h because the cell increases the transcription gene as much as possible. As a logic consequence the siRNA is exhausted very fast due to the strong increased amount of matching transcripts.

When the gene is very essential the population of apototic cells will be very high ~50% (FACS analysis in my case) and the cells you harvest for your RNA extraction are cells which have survived because they only get very low amounts of siRNA --> no knock down and this population falsifys your gene expression results.

In my case I have 95% growth reduction after 72 hour incubation. FACS analysis showed me 60% of apoptotic cell which mostly folats after 72 hours. After 48 hours I have an expression level of 70% and after 24 hours of 50% due to the rapid apoptosis induction. So I can not determine the epxression level exactly because  apoptotic cells are washed away where a good silencing took place and only the surviving fraction with a weak knock down is harvested by RNA extraction and responsible for the weak knock down results. 

I would try to determine the knock down after 24 and 48 hours with RT-qPCR, there you should see an effect and also that the expression increases from 24 to 48 hours. If you want a longer lasting effect you can use the 10 fold amount of siRNA but first try to determine the expression level after 24 and 48 hours if you see an effect. But if you see a reduced protein level after 72 hours you can also make western blots for 24 and 48 hours to show your successful knock down and some reviewers like protein reduction more than mRNA knock down.

Regards

Hello Arnab,

An simple explanation is, if the gene/protein is very essential for the cell type you are using, then you see a decrease in the protein level after 72h but the RNA level is back at the normal level after 72h because the cell increases the transcription gene as much as possible. As a logic consequence the siRNA is exhausted very fast due to the strong increased amount of matching transcripts.

When the gene is very essential the population of apototic cells will be very high ~50% (FACS analysis in my case) and the cells you harvest for your RNA extraction are cells which have survived because they only get very low amounts of siRNA --> no knock down and this population falsifys your gene expression results.

In my case I have 95% growth reduction after 72 hour incubation. FACS analysis showed me 60% of apoptotic cell which mostly folats after 72 hours. After 48 hours I have an expression level of 70% and after 24 hours of 50% due to the rapid apoptosis induction. So I can not determine the epxression level exactly because  apoptotic cells are washed away where a good silencing took place and only the surviving fraction with a weak knock down is harvested by RNA extraction and responsible for the weak knock down results. 

I would try to determine the knock down after 24 and 48 hours with RT-qPCR, there you should see an effect and also that the expression increases from 24 to 48 hours. If you want a longer lasting effect you can use the 10 fold amount of siRNA but first try to determine the expression level after 24 and 48 hours if you see an effect. But if you see a reduced protein level after 72 hours you can also make western blots for 24 and 48 hours to show your successful knock down and some reviewers like protein reduction more than mRNA knock down.

Regards

Dear  Arnab

( I have NOT used DHARMACON products)  On a hindsight, is the product specific to any ShRNA expression?

Even if itw ere the case, I wonder if you could expect significant control of events for two levels.  Would you?

Best

Prash

PS:  Also, please await for more answers from experts.  

Hi Arnab,

I would suggest to see the effect on RNA level at lower time points. If you observe the effect  at the protein level, after 72 hour, try to follow at lower time point,  a time kinetics can be done so that you can see the differential expression  at the RNA level.

Sanjay

Dear All,

Thank you for the comments.

Though I had tried to look into the RNA levels for both 48 and 72 hrs, with no alteration, I have not tried looking into lower time points. I will try out for the lower time points for the proteins too.

Am open to further comments.

Although I often see a reduction in mRNA levels with siRNA or shRNA, one recognized mechanism of miRNA inhibition of gene expression is, if the miRNA matches completely to the target sequence, it can inhibit entry into the ribosome without inducing degradation of the mRNA.

Although I must say, I do like Volker's answer. I have found it quite difficult to knock down MYC in aggressive cancer cells, because it is such a fundamental protein and regulates transcription itself. As we normalize by protein quantity, we might not see a difference in levels of proteins that themselves regulates transcription and/or translation. So, in addition to what Volker suggests, perform the transfection, check the health and number of your cells at those time points. Secondly, if you have a good reason to do so, you could normalize by the number of cells at time of harvesting, rather than RNA or protein quantity. 

Let us know how it works out!

siRNAs and miRs can affect expression by either inducing mRNA degradation or by preventing translation. It could the that what is being repressed is translation of the message, but not its actual abundance/concentration. That would only affect protein abundance but not mRNA concentration.