Well, who knows, might actually stabilize lncRNA through base pairing. In fact, many natural antisense transcripts stabilize their sense counterparts. Actually, Lincode siRNA reagents from Dharmacon may prove efficient in knocking down long noncoding RNA. And, of course, these days we could relatively easy use for this purpose CRISPR interference.

thankyou @igor

we could have considered the CRISPR interference but the experiment is already established and stable, repeated 3-4 times before,,i couldn't figure out why i am getting this opposite effect now.

Hi Syeda,

I can find at least one of possible reason of such effect. As I understand, you have prepared the new primary culture, and not the same used by previous researchers?What is the score of overexpression? Note, that lncRNA's expression is may be case depended. Try to ask your colleagues if any cDNA from the previouse tests is left, and use it as double referenc control.

Thanks for your response Andrey.

yes i had prepared the new primary culture,, but it was similar like they had used, i mean the cardiomyoctyes from neonatal KM mice.

can you please explain what do you mean by ''lncRNA's expression may be case depended'' ??

i'll try to get  the cDNA from others, but they had successfully knockdown the lncRNA, so how would that help me to know the problem?

Hello Syeda,

First I have a question concerning the siRNA you are using, it is a single siRNA sequence or a pool of several sequences?

If it is only one sequence you can have the following problem when you have prepared a new cell batch from your KM mice. I do not know if this mouse strain is compleately homogenous in its genetics, which means every mouse has an identical genome like it is in classic inbreed mouse strains. If they are very different in their genetics, like outbread mous strains, you can face the problem that your new cells, you are using, have a SNPs in the siRNA binding area and disturbing your siRNA/lncRNA duplex formation and the RISC complex is not cleaving the duplex anymore and no silencing occurs. That would be a pity but possible if the cells are genetically heterogenous.

In fact it could also be that your transfection is not working well. I have made the experience that the lipofectamine can lose its function after a while and so I have prepared aliquots that every month it is used up. Try a new Lipfectamine and have a look if the result turn better.

At least I have also detected that important genes for a cell undergo a strong up-regulation of their transcription by transcription factor due to the fact that the cell simply needs them to survive. When your siRNA is not equipped with chem. modifications preventing it from intercellular nuclease activity your siRNA is degraded during your incubation time. I have seen no knock down after 48h, but a very strong one after 24 h in my experiments and in both time points the transcription factor of my gene of interest was increased 2-3 fold simply exhausting my silencing effect over time. In this case only chem. mod. siRNA improves the effect or prolongs the knock down.

It could also be if your siRNA is natural without chem. mod. for stabilization, is mechanically degarded when you very often that it freez thaw your vial. When you polute your siRNA a bit and there are then nucleases in your vial you end up only with nucleotides after several times thawing your tube - no effect. Also prepare working aliquots and store the concentrated stock at -70°C. Working aliquots at -20°C. The working aliquot should be used up after 5-10 transfections.

How long do you incubate your cells and is the lncRNA important for your cells, then your waiting time is maybe too long?

I hope this suggest help you a bit :-)

Regards

i had prepared the new primary culture, but it was similar like they had used, i mean the cardiomyoctyes from neonatal KM mice.

KM mices are not identical (see Volker's comments). That what i mean by "case depended".

i'll try to get  the cDNA from others, but they had successfully knockdown the lncRNA, so how would that help me to know the problem?

use your own cDNA as sample and cDNA from others as reference: may be your housekeeping genes are downregulated.