Can RNAi cause RNA levels to increase?


We’re trying to confirm if feeding RNAi is decreasing a specific gene. We’ve taken the precaution of designing one primer in the 3’UTR and the RNA was DNAse treated to limit genomic DNA contamination. The targeted gene cDNA is actually increased by about 3-fold. We did -RT controls to be sure and they have no amplification. I also know that the primers are not picking up the feeding plasmid, because one binds in the 3’UTR and we get no amplification with the plasmid as a template.

I’ve heard of RNAi mediated RNA polymerase, but I thought that this only occurred in the 3 to 5’ direction. Has anyone experienced or heard of this before? I should mention that the strain is TU3335 lin-15B(n744) X; uIs57 [unc-119p::YFP + unc-119p::sid-1 + mec-6p::mec-6], which was made to be RNAi sensitive in neurons and is somewhat refractory in other tissues.

Any insight would be greatly appreciated.


It could be something specific about that particular gene.
Which RNAi clone are you using? Which gene are you targeting?

PD: I assume that the other primer binds to a exon-exon junction

The gene is anoh-1 and it is expressed at low levels in whole worms (via RNAseq counts) and mostly in neurons (via reporters).

bearing in mind that I am no expert in RNAi, is it possible you are seeing a block on anoh-1 translation and (via a feedback loop) an increase in transcription?

I do not see any 3’UTR sequence for the two transcripts of this gene. According to wormbase, there is only a 5’ UTR in the shorter transcript.
At any case, I guess that you are using the RNAi clone for the ORFeome library, which was made from the cDNA of the shorter transcript.
To avoid the possibility of amplify by RT-PCR the sequence for the RNAi clone, I would use the clone from the JA library or I’ll made a new clone, using later RT-PCR primers out of the RNAi targeted regions to check for the RNAi efficiency.

When I look at RNAseq reads, there is a short 3’UTR and that’s where I put my reverse primer. I think it’s not annotated because there is a repeat region in it that I’m guessing interferes with the mapping of reads. The RNAi clone is one that we made from start to stop codon, so that is why I put one of the primers in the 3’UTR. If I use the feeding plasmid as template, I get no product, so I’m pretty confident that I’m not getting amplification of the plasmid in my worm samples.

Hi kchoe,

My guess is what you are seeing is a PCR artifact. No matter how much you wash your worms, your sample is likely contaminated with the RNAi bacteria. These bugs are brimming with dsRNA for your target gene. After RT, then, you have a mixed population of cDNAs, some derived from your gene of interest and some derived from the bacterial dsRNA. Such overlapping DNA fragments can be fused during PCR (see fig 2). So I bet that you are detecting a fused PCR product between your worm derived cDNA and the bacterial derived cDNA. For this to be the case, the dsRNA have to be abundant enough to make it through the RT step, which I guess they probably are. You might consider an alternative method for measuring RNA levels. Maybe make a smaller RNAi clone and do a northern?


Thanks Sven,

I think your idea makes sense. I was having a hard time thinking of other potential artifacts that I had not ruled out and the idea you mention makes sense. I think I’ll try silencing a translational reporter next.