Actually, I don't think this is particularly unusual.  I've seen similar things with very small volumes of liquid, including cDNA reactions, even after overnight in the freezer.  I might have a strip tube with similar reactions, six or seven will look frozen, but one or two will look liquid.  Freezing into ice is a crystallization reaction and crystallization requires "nucleation'.  In very small volumes, the probability of a 'nucleation" event may be reduced.   Sometimes, if you give a frozen tube that still looks liquid a good tap, it may crystallize--the vibration induces a crystallization.  In any case, unless and until you actually have a  functional problem with downstream reactions showing anomalies, I would not worry-its just the wonder of nature...

hi dear 

may be you need further purification and further washing steps to get rid off the salt residues (from buffers or so in your kit) since theses salt residues lower the freezing point of your samples

Sounds like it could be high salt, which could be problematic. Ethanol precip. should help

Dear Dario

if you are using the classical method for extraction the DNA , then ethanol or isoamyl alcohol will be  good choices , but there is an isolation-purification kit for DNA which contains silica for purification which adsorb the salts and let your purified DNA moves out of the column .

Actually, I don't think this is particularly unusual.  I've seen similar things with very small volumes of liquid, including cDNA reactions, even after overnight in the freezer.  I might have a strip tube with similar reactions, six or seven will look frozen, but one or two will look liquid.  Freezing into ice is a crystallization reaction and crystallization requires "nucleation'.  In very small volumes, the probability of a 'nucleation" event may be reduced.   Sometimes, if you give a frozen tube that still looks liquid a good tap, it may crystallize--the vibration induces a crystallization.  In any case, unless and until you actually have a  functional problem with downstream reactions showing anomalies, I would not worry-its just the wonder of nature...

I agree with David. The viscosity of cDNA however raises a red flag to me. I have never noticed this. Is it possible that you have DNA not cDNA in your sample? even DNA shouldn't be noticeably viscous. 

Thanks Meelad, do you have any particular kit that you may recommend? I have a purelink dna isolation columns for gDNA isolation. Dont know if it is any good for this application...please let me know if one in particular works best for you. Today I ran a qPCR experiment with those cDNAs and the efficiency for the standard curve was 60%. I assume a possible reason could be that salt contamination. Any comment?

Dear Dario

The kit you're using  for gDNA extraction sounds good , i usually deal with Promega products for extraction and they gave me convenient results .

When you say that the samples are viscous, how are you assessing this?  Are all of the samples viscous?

Was there any variation in the apparent PCR efficiency in the different samples?  You can make a rough assessment of this by using a logarithmic display and looking at the (early cycle) slopes of the curves in different samples for each primer pair tested.  If there are noticeable slope differences between samples, then there is some source of sample variation.  This might be due to different levels of some contaminant in the RNA preps or it might be due to insufficient mixing of your RT master mix when you set up the reactions to make cDNA.  Since the RT enzyme has glycerol in it, it will sink to  the bottom of the tube.  If not mixed properly, this will cause different samples to have very different amounts of enzyme and glycerol.  Variations in the amount of glycerol would cause differences in freezing temperature and differences in the viscosity of different samples.

Also, how did you calculate the efficiency of the reaction with the standard curve?  Some details on this and a graph of the results might be helpful.

Dear David, thanks for your reply. I am not entirely sure if this is the answer you were looking for, but I will try anyway. I calculated the standard curve by adjusting the threshold directly on the Aria MX software. The best threshold setting gave an efficiency of 61% (I cannot remember the slope at the moment, but will provide that info tomorrow). I will try again with different cDNA preps and determine if there is any difference, but from the comparative run that I performed after the standard curve I did not have any major issues. I mixed the RT well before use, but I will definitely keep an eye on that, Also my RNA preps were pure (260/280=2; 260/230= 2.1) and with good integrity (RIN=10). Most of the other cDNAs did not have that problem anymore and even the viscous one did freeze after a day. I have only assessed the viscosity by observation and direct comparison with the RT's buffer viscosity. That is why I got worried that I may had contaminants. I have attached a copy of the report on that run. Unfortunately I dont have access to all the data from home, but please have a look at this, if helpful. I will provide more info tomorrow. Thank you very much for the support.

Ok, it looks like this sample had some PCR inhibitor in it.  I'm not sure what dilutions you made, but if the same amount for each point, (that is 1/4 or 1/3 serial dilutions), then it looks like the inhibitor is getting diluted out and the Ct increase that you see with each dilution is much less than what you would expect from the size of that dilution.  (If the PCR efficiency was actually poor, then the spacing between the curves for each dilution would be about equal, but it would be a larger spacing than expected from an efficient PCR. ) But what you are seeing is unequal spacing--that is a very small Ct increase with high amounts of template and increasing Ct differences with lower amounts of template, which is more consistent with an inhibitor.  (I am a bit perplexed that the shapes of the curves are not more different though. Is my assumption that these were serial dilutions correct?)