the problem was that I loaded 1 micro litter in 2 two different wells which contain the same mix and also the same cDNA and one of them give good signal and the other doesn't.

Ok, you've loaded your plate quite confusingly (normal convention is to put replicate wells in a row, rather than distributed over the plate), but I can see where your problems lie.

Note: your Cq values are HIGH. High corresponds either to low [cDNA] or to poor PCR efficiency. GAPDH should not be giving Cqs of 25-28: GAPDH is a very robustly expressed gene, and I would expect something more along the lines of 16-20.

As a consequence of this, for many of your other genes (which are presumably not so robustly expressed), the Cq values are in the mid 30s: as a rule of thumb, a Cq of ~35 corresponds to a single template molecule, so you are, as Mikael Kubista

An irritating aspect of qPCR is that cDNA synthesis buffer itself can inhibit PCR reactions, thus you might see the things you're showing if you have

A) far too little cDNA,

but also

B) far too MUCH cDNA.

I.e. if you put LOADS of cDNA in the well, you get very high Cq values purely because the reaction is barely occurring.

So, why not provide us with a bit more detail regarding your experimental set up and we'll see if we can troubleshoot this for you?

The above possible reason is very well mentioned. It may sound very weird & non-scientific reply but could be technical.

we also something get results like "UNDETERMINED", actually we found that was the issue of qPCR instrument calibration.

John Hildyard Thank you

When I did reverse transcription reaction I made sure to load just 500 ng of the RNA sample then as the kit recommended 30 Micro-litter Nuclease free water to the product. I prepared a master mix for 13 sample for each gene, as the last one is for pipetting error issue, after this I load 19 Microlitter in each well and 1 Microlitter cDNA

I run the reaction as the protocol provided by the kit.

is it possible that the amount of cDNA is low? If I add 2 microlitter for each well does it really matter ??

Saurabh Mandal

I don't think it's the instrument calibration because my friend use ready to use plate he just add the cDNA and the instrument work fine.

Ok, let's troubleshoot: how are you isolating RNA, and from what samples?

How are you measuring [RNA], and if nanodrop or similar, what are your 260/230 ratios like?

What cDNA synthesis kit are you using, and when do you add your 30ul of nuclease free water (and why)?

John Hildyard

I extracted RNA samples from liver and white adipose tissue using

RNeasy Mini Kit (Qiagen) I run gel elctrophoresis for the RNA samples. the bands was very good and show RNA.

then Nanodrop and the concentration was good enough some samples 200 ng and others 500 ng.

The 260/230 ration as provided by the attached pic.

I add appropriate amount of RNA to the reverse transcription so that I will have 500 ng of the RNA in each tube.

I use to cDNA synthesis kits (Qiagen and Intron)

I added 30ul of nuclease free water as recommended by Intron kit but not Qiagen.

they add this amount of N.F.W to dilute the cDNA sample and so it will be suitable for RT-PCR.

the problem of getting unknown Cq for some duplicate when I try both kits but in each time I run RT-PCR I just add 1ul of cDNA.

The most obvious problem here is you have salty RNA.

The 260/280 ratio determines protein contamination: all your values are ~2.00, which means you have essentially no protein contamination.

The 260/230 ratio determines salt contamination (specifically, the chaotropic guanidium salts that virtually all RNA isolation methods use). You want these to be around 2.00 as well (though I usually accept anything greater than 1.70).

Low ratios here mean that when you add your RNA to the cDNA synthesis reaction, you're also adding a lot of salts that unfold/inhibit proteins, so your cDNA synthesis efficiency is likely to drop right down. This would explain why your Cq values are quite high: you're simply not converting much of your RNA to cDNA.

Thankfully, RNA is pretty easy to clean up: I typically just reprecipitate it:

Add DEPC/Nuclease-free H2O to your RNA to make the volume 500ul

Add 50ul of 3M sodium acetate, pH 5.5

Add 10ug of glycogen (optional, increases yield)

Mix well

Add 500ul room temperature isopropanol.

Mix well, incubate at room temp for ~20mins, spin at max speed in a benchtop centrifuge (chilled if you have one) for 10-15mins, wash 2x with ice cold 75% ethanol, air dry, resuspend.

This process will usually lose ~20-30% of your RNA (you'll retain more if you include glycogen), but will remove the vast majority of your salt contamination, leaving the remaining RNA much more cDNA-synthesis friendly.

John Hildyard

Thank you so much I was thinking about 260/230 ratio that the ratio was very low which is not as recommended by the extraction kit but I didn't know what does that mean.

I will try the RNA clean up method.