RNAi validation

Hi all,

I’m having trouble to measure the levels of expression of a gene interfered by RNAi. I have repeated the RT-PCR three times and the levels of expression of
the interfered strain are higher than those of the wild strain. I have tried several primers of the gene and several housekeeping genes, but the
results are the same. Also, I´ve tried primers in the 3´UTR, more washes and purification by poly-A. Apparently i can´t get rid of the plasmid, does anyone know if the plasmid L4440 has a polyA tail ???

Hi Yamila,
3’UTR primers work only with RNAi clones derived from the C. elegans ORFeome Library v1.1 (http://www.thermoscientificbio.com/uploadedFiles/Resources/c-elegans-orf-technical-manual.pdf).
Just get it, and you will have no problem anymore with knock-down verification.

Cheers, oldcoffee

Hi oldcoffee,

Thanks for your answer. the RNAi clone is from thermoscientific but the 3´UTR of my gene is really short so i can´t measure the expression. I did poly-A purification but it doesn´t work (the levels of the interfered strain are high). So i was wondering if the plasmid from thermoscintific has a poly-A tail. (it isn´t specified in the data sheet)

Many years ago, we tried to RNAi unc-15, bleached worms to get embryos, used RT-PCR to detect unc-15, and also observed an increase of unc-15 mRNA. Later, we thought that for unc-15 worms, there is a “bag” phenotype. Therefore, the embryos from animals after RNAi had a different stage distribution with those of control animals.
Maybe this should also be considered.

good luck.

Thank you but i am not sure if a I understood what you mean. you say that may be i am measuring the ARNm of the adults and eggs inside de animals?
in each condition, when i do the RT-PCR, i use a plate with worms of all the stages (eggs, larva, adults)
in the interfered animals i see a fenotype, like reduced lifespan and egg laying compare to de wild type.

Comparing gene expression from mixed population can be troublesome, since samples can have different composition of each stage. It is much better to use synchronized animals.

Hi Yamila,
i had the same problem like you. You can’t get rid of the RNAi plasmid. You need to design primers that bind in the 3’utr or the 5’utr. It’s ok when only one primer of the pair binds in these regions.

Which gene do you knock down? I will look it up, whether such a primer design is possible.

Cheers, oldcoffee


I have tried 3´UTR but the primers did not work out because the sequence is rich in A-T. The gene is nmgp-1 .
To measure the expression I normalize the cuantification with housekeeping genes and synchronize the animals
I can see alterations when this gene is interfered, so i know that it is working but i can´t validate the interference by RT-PCR.

Thanks for your help!!!


Hi Yamila,
Primer design is possible in the 5’utr:

For real-time PCR:
Amplified product size: 116 bps

For conventional PCR (agarose gel):
Amplified product size: 501 bps

I recommend using random hexanucleotide primers for cDNA synthesis, as the amplified product is in the 5’utr region. But polydT primer will probably also work because transcript is not large.

I promise, it will work.

Cheers, oldcoffee

Thank you!!! I will try it!

Cheers, Yamila