On 2017 Oct 01, Misha Koksharov commented:

Of interest, depletion of an individual MRTF isoform (either MRTF-A or MRTF-B) almost completely suppressed induction of several contractile proteins despite the presence of the other MRTF isoform. This most likely indicates that both isoforms cooperate in expressional up-regulation of contractile proteins and that a certain threshold concentration of MRTF is required in order to stimulate the EMyT.

During my previous postdoc I've observed similar effects of silencing MRTF-A and MRTF-B separately in U2OS cells when using bioluminescent real-time reporters ("SRF-luciferase") as an output. Before me it was also noticed by Gerber et al, 2013: Fig. 4. We have used the same siRNA SmartPools from Dharmacon: (siRNA sequences are in the links) against human MRTF-A, MRTF-B and SRF (and in some experiments - against mouse SRF).

However, after a more close investigation with individual siRNAs from these pools, I came to a conclusion that the observed necessity of both MRTF forms was caused by off-target effects of some of the individual siRNAs (at least in my system):

a) Three of the MRTF-A siRNAs also reduced mRNA levels of MRTF-B. Only one was true anti-MRTF-A siRNA (and this one also gave the highest reduction of MRTF-A mRNA levels) but it didn't reduce the reporter induction in U2OS cells in marked contrast to the pool.

b) One of the anti-MRTF-B siRNAs caused some general off-target effects in addtion to MRTF-B knockdown: considerable cell death, blocked not only SRF but also some other pathways. Its effect on SRF-luc persisted even when the target site in MRTF-B was mutated to prevent the targeted silencing. Three other siRNA prevented most of the SRF reporters's induction and it was reduced further by adding the good single anti-MRTF-A siRNA.

The overall conclusion was that at least in U2OS cells the MRTF-A is not essential in these reporter settings but shows a minor partial contribution if MRTF-B is depleted.

I'll probably include this data in the later paper but for now I hope these notes will be helpful for people using these MRTF/SRF SmartPools. I strongly recommend to check the effects of individual siRNAs in them.

By the way, one of the siRNAs in human/mouse SRF pools also showed noticeable off-target cellular toxicity (and it was not even the strongest in reducing SRF mRNA levels). Given that each of the 3 pools I've worked with had some kind of off-target effect, this confirms the warnings of Jackson AL, 2010 that this can happen quite often. :( Thus, it seems that in general it is highly desirable to check all the individual siRNAs in commercial pools for target/off-target effect and have at least 2-3 different siRNAs against the same gene confirming the knockdown phenotype.

On 2017 Oct 01, Misha Koksharov commented:

Of interest, depletion of an individual MRTF isoform (either MRTF-A or MRTF-B) almost completely suppressed induction of several contractile proteins despite the presence of the other MRTF isoform. This most likely indicates that both isoforms cooperate in expressional up-regulation of contractile proteins and that a certain threshold concentration of MRTF is required in order to stimulate the EMyT.

During my previous postdoc I've observed similar effects of silencing MRTF-A and MRTF-B separately in U2OS cells when using bioluminescent real-time reporters ("SRF-luciferase") as an output. Before me it was also noticed by Gerber et al, 2013: Fig. 4. We have used the same siRNA SmartPools from Dharmacon: (siRNA sequences are in the links) against human MRTF-A, MRTF-B and SRF (and in some experiments - against mouse SRF).

However, after a more close investigation with individual siRNAs from these pools, I came to a conclusion that the observed necessity of both MRTF forms was caused by off-target effects of some of the individual siRNAs (at least in my system):

a) Three of the MRTF-A siRNAs also reduced mRNA levels of MRTF-B. Only one was true anti-MRTF-A siRNA (and this one also gave the highest reduction of MRTF-A mRNA levels) but it didn't reduce the reporter induction in U2OS cells in marked contrast to the pool.

b) One of the anti-MRTF-B siRNAs caused some general off-target effects in addtion to MRTF-B knockdown: considerable cell death, blocked not only SRF but also some other pathways. Its effect on SRF-luc persisted even when the target site in MRTF-B was mutated to prevent the targeted silencing. Three other siRNA prevented most of the SRF reporters's induction and it was reduced further by adding the good single anti-MRTF-A siRNA.

The overall conclusion was that at least in U2OS cells the MRTF-A is not essential in these reporter settings but shows a minor partial contribution if MRTF-B is depleted.

I'll probably include this data in the later paper but for now I hope these notes will be helpful for people using these MRTF/SRF SmartPools. I strongly recommend to check the effects of individual siRNAs in them.

By the way, one of the siRNAs in human/mouse SRF pools also showed noticeable off-target cellular toxicity (and it was not even the strongest in reducing SRF mRNA levels). Given that each of the 3 pools I've worked with had some kind of off-target effect, this confirms the warnings of Jackson AL, 2010 that this can happen quite often. :( Thus, it seems that in general it is highly desirable to check all the individual siRNAs in commercial pools for target/off-target effect and have at least 2-3 different siRNAs against the same gene confirming the knockdown phenotype.