Firstly: how certain are you that it is actually being expressed? If it's simply not there, you're not going to see it no matter how concentrated you make your RNA.

Secondly: You don't state how much cDNA you are using per well. You're using 1ug of RNA for cDNA synthesis, but how much of that are you using in each well (assuming 1:1 conversion)? What volume is your cDNA in? What volumes are your PCRs?

Thanks for your answer. My PCR is 12.5 microliter and I am using 2.5 microliter of cDNA. I am sure that it is expressed because I am able to silence it. When I silence, the phenotype is changing.

I would suggest trying with different primers.

2.5ul of neat cDNA?

Assuming your cDNA synthesis is in 10-20ul vols (or thereabouts) this is far, far too much cDNA, and your reactions will most likely be markedly inhibited by cDNA synthesis buffer (or simply vast excess of potential primer binding sites).

I would dilute your cDNA down perhaps 10 fold, then use 2.5ul of this.

For reference, I typically use 800ng of RNA, in a 10ul cDNA reaction, which I then dilute 1/20 to give final [cDNA] (assuming 1:1 conversion) of ~4ng.ul-1.

I use 2ul of this per well, 10ul vol (so ~8ng of cDNA per well) and this suffices even for very low expression targets.

As for your silencing: having an effect on the phenotype is not a priori evidence your lncRNA is expressed, and indeed, without a means of quantifying expression, you cannot even claim you ARE silencing it.

It is certainly possible, but depending on your chosen method of gene silencing, there are a number of other possible explanations.

In the absence of a suitable positive & negative controls, I would caution against over-interpretation.

A Cq of 11 is considerably too much target material in the well.

Thank you all for your answers. The cDNA synthesis reaction is 20 uL and I am adding 30 uL of water at the end of the reaction as the protocol says. I didn't know that I should dilute it more. Actin and GAPDH Cq values are 15-16. I think if the cDNA synthesis buffer inhibits the qPCR reaction, I would not have expression GAPDH and actin. Maybe there is something wrong with my primers.

I also think that Cq 11 is too much and I thought maybe I used a lot more plasmid for overexpression than I should. Because it was my first tiral and now I am trying different dosages. But can I trust the primers now or should I design new ones ?

A quick test for cDNA inhibition of qPCR: dilution series.

If you take your cDNA and use it to make a 10-fold dilution series, then run these (for something high-expression like GAPDH). If all is well, you should see your Cq values increase by ~3.3 with each dilution, up until you hit Cq 30 or so, where it should become increasingly erratic (as at these dilutions you're measuring tens of transcripts, so stochastic partitioning effects come into play).

If you have cDNA inhibition, you may instead see the Cq values stay similar for the first few dilutions (or even decrease), before eventually settling into a nice linear relationship: this represents the high [cDNA] containing more transcript, but also more inhibitory components, giving you a non-quantitative reading.

Ideally you want your cDNA to be as dilute as you can get it while still allowing you to collect meaningful quantitative data from all your samples.

Thank you very much! I will try this as soon as possible

Try using more than 2.5ul of cDNA in your reaction...eliminate H20 and replace with cDNA.

can we use random hexamer primer for cDNA synthesis of lncRNA?

Dear Dilek, just curious if you solved your problem? Did diluting your cDNA work? I am facing a similar problem. I cannot detect the expression of my gene in certain samples but got a Ct value around 20 for my over-expressed sample

Dear Arpita,

I solved the problem by changing the cDNA synthesis kit and primers. My primers' product length was about 250bp. They work fine with normal PCR but did not with qPCR. I switched primers with another pair which is designed by qiagen company and the product length is 90bp. Our previous cDNA synthesis kit allows to use only 1ug of RNA, we switched to Thermo cDNA synthesis kit which allows up to 5ug RNA that yields higher concentration. Now my qpcr results are quite fine.

Thanks very much for the answer Dilek. Another question though- you had said that you obtained Cq value around 11 for overexpressed sample which means your old primers did work in the Q-PCR.Am I getting this right? My Q-PCR primers amplify a product around 75bp and I obtained a Cq around 20 for my positive control over-expressed sample, so I think my primers are alright. I still couldn't get overexpression in my test samples however. How much RNA are you converting to cDNA now, with this new kit?

Dear Arpita,

Since the lncRNA are expressed generally at lower levels, and detection of these RNAs becomes tedious and difficult. The best way, I think, is to check the primers for lncRNAs using a series of dilutions of cDNA from 1:10 to 1:1000 or more. Also, do a qPCR reaction of template only, primer only samples. The qPCR reactions once loaded on TBE-Acryamide gel will tell you the right dilutions and the non-specific amplification from the primers also.