i extracted RNA from MCF-10 by trizol , and ct of cDNA of MCF-10 of LKB1 gene gave strange value ..cDNA diluted 1:4 ,ct value with GAPDH 11 in triplicate, but with interested gene ct values different in triplicate 38, 42 and 48 and different melting curve and i repeated the experiment gave me the same value..
although when i diluted the sample 1:20 ,1:50 not gave me any expression with LKB1 gene ..and with mcf-7 cell ct value for interest gene gave good ct at 28 ..lkb1 is tumor suppressor and should decrease in mcf-7 and increase in mcf-10 and that not happened with my results
hi,
Ct values above 35 are usually considered negative, meaning no or almost no transcript present in your initial sample. when your specific cDNA is in very few copies you may assist to random sampling of 1, 2 or no copies for each tube, giving rise to very diverse Ct...
About the different melting curves, have you checked if they are due to primer dimers or hairpins? try using positive (cell lines or conditions where your gene is definitely expressed) and negative (no template: this will allow, among others to check the T melting of primers) controls
Good luck
Hi Asma Future,
Do you use nanodrop for your calculation to find out required amount of cDNA per Real time PCR ? . If so, I want to explain one of controversial issue about nanodrop. Nanodrop is actually not effective to distinguish concentration of single strand DNA (single strand RNA is converted into single strand DNA in cdna reaction) and single strand RNA. If you make a cDNA with a different brand then your calculation about concentration will be different since the effectiveness of those different brand are not same. Since nanodrop will also give a controversial concentration about your samples it will affect your RT-PCR results
So I suggest you to put same amount of RNA amount per cdna synthesis and to use same brand of cDNA kit for following RT-PCR. Also only consider the initial concentration of added RNA product into cDNA reaction for your RT-PCR calculation.
I hope to be helpful for you. Good luck.
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