Hi Rajesh

The amplification plots are looking like a 'Raw data' to me ...are you sure it is really processed after the PCR run...Or else there is something wrong with your initial sample tube..if you see the initial fluorescence (at 0 time) of your problematic sample it is well below the other tubes.....how ever the amplification pattern of that sample is similar to other tubes.....appears to me something in quencing the fluorescence in the first tube.....and a careful analysis show that the Ct of this tube is lower than the next serial dilution...

Hello Ajay,

I am sorry, I do not understand what do you mean by raw data. In fact, I did serial dilution and run the pcr reaction with the telomere primer. The first dilution have no Ct. When I viewed the text result, it doesn't show any Ct values. I tried to run the sample doing another serial dilution from the same stock, but again I got the same result. The results showed that it has got either no Ct value or higher Ct value than the dilutions following it. I do not understand how can it be possible -- I mean, the higher the concentration, the lower should be the Ct value.

And even this amplification curve looks weird to me which has not even crossed the threshold level and then went down.

Hi Rajesh

The 'Raw' and 'Processed' data I was mentioning is about the way it is shown in 'Eppendorf' real time PCR machine...So I enquired if you have shown that data....But I will suggest you to address following points

1) You should have taken more range of dilution ....the range sould have such that you Ct of the gene should ideally be between 15 to 30-35.......The Ct values indicate that your sample have more cDNA...so you should further dilute it

2) The 'no Ct' you are mentioning for your 'first concentrated sample' is based on the automatic threshold set by the machine/sofware.......if you care fully see the plot ...you can always adjust this value manually and can see that your software will give Ct for this sample also.......

3) Don't always go for results based on SYBR based 'amplification plot' only.....it is not indicative of your gene of interest....it shows the amount of total DNA in the tube....whether it is your 'gene' or 'some other band' or 'primer dimers' or 'combination of them',,,,,,,always link this data with the 'Melting Curve' analysis data.....

4) Verify your results on agarose gel initially for few times and compare it with the real-time data...

5) After looking at you plots.....in my opinion...your sample 1 (concentrated sample) and sample 6 (sybr + primer, if I am correct) have some problem...they are showing almost similar profile. but much less fluorescence compared to other samples..but only one is showing Ct...Both can show this with some manual intervention in setting the Threshold value.....but I will suggest you to compare their 'Melting Curve ' with other samples..and also on the gel too...so get insight into the problem..

6) You NTC is also givng an amplification plot with early Ct (30.84)....if it is giviing simlar melting curve ...it is indicative of template contamination.....so check your reagents..

address these concerns and redo experiment and analyze the results again.....

all the best

bye

Thank you Ajay for your kind suggestion. I will address those things. I have tried to dilute my sample (the standard) that I have ordered from the company. I am getting another problem now. I have just posted one more question in PCR topic. Can you please check it? I want your valuable advice and guidance.

Thank you so much once again for answering my questions.

A bit late in answering, but it looks to me like they have not been normalized.