the qPCR standard curve for a B.bifidium DNA starting from 20ng to 0.00002ng is giving strange Ct values , eventhough i repeated the qPCR three times in case i mixed up the serial dilution tubes. The Ct value for the highest conc DNA is higher than the less concentrated !What could be the reason? Knowing that the efficiency of the reaction is 90.2%. Below are the average Ct values i got .
20ng---avg Ct=20.37
2ng----avg Ct=16.06
0.2ng----avg Ct=19.02
0.002ng----avg Ct=23.45
0.0002ng---avg Ct=31.29
0.0000ng----avg Ct=35.56
Hi,
very probably you have some inibithory. So when you diluite the template, these sostances do not interfere with your taq. But if you diluite a lot your template, you have not template to start the rt-pcr.
How do you extract your template?
I hope to be clear. Best. Olga
Hi,
very probably you have some inibithory. So when you diluite the template, these sostances do not interfere with your taq. But if you diluite a lot your template, you have not template to start the rt-pcr.
How do you extract your template?
I hope to be clear. Best. Olga
Olga is most likely right. Your 20 ng sample probably contains inhibitors that are diluted out in higher dilutions. But do check on the background subtraction. If it set to cycles 3 - 15 (standard) it will distort the 20ng (and probably the 2ng) curves. This will make them look anomalously flat.
Incidentally, you may have made an error listing the amounts. Your results would make much more sense if the series is 20 - 2 - 0.2, 0.02, 0.0002. Otherwise the jump from 19 to 23.4 is too small and the jump from 23.4 to 31.3 is too large.
Dr. Andrew, the amounts listed are right and i was actually wondering the large jump from 23.4 to 31.3 and the reason behind it, knowing that i got the same results of Ct using different aliquouts of B.bifidium DNA 10-fold serial dilution.!!! As a result of this standard curve , what should i do in order to be able to quantify Bifidium in my samples ??? Can i depend on the curve starting from 2ng to 0.002 ng only????
Thanks for your answer Olga ......My template is B.bifidium 24hr culture that was extracted by a QIAGEN DNA isolation kit. How can i get a better standard curve without going back to culturing and DNA isolation ??
I think that you can purify again your DNA. I have tried the Glass Milk for my templates from soils and the results were good.
In first istance, I used a good kit for soil, but however I had your same problems, so I have used also Glass Milk, i.e.:
the obtained DNA was re-purified using GeneClean II Kit (Qbiogene, MP Biomedicals) following the manufacturer’s protocol.
I hope it is useful for your templates.
Ciao, Olga
First I would ask you if you know MIQE (Bustin 2009, Clinical Chemistry 55:4
611–622 (2009). They speak of a convention in terminology on real time PCR in the name of many editors of qPCR publishing journals. The term Ct must be replaced by Cq. I suggest you to read that paper!.
Second, I would like to see the amplification curves graphed logartihmically and tho see the intercept between succesive concnetration. You see a big gap between the Cq data at 0.002 and 0.0002. I think that you only make non- specific products at the latter dilution. The more that in the blanc you find a Cq as well. Inhibition is likely at the highest concnetration. Al this indicates that your qPCR is suboptimal
Thanks Elizabeth for your notes .Attached the amplification curves of the standard 10-fold dilutions from 20ng to 0.00002ng
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