Hello everyone
I have a problem with real-time PCR.
My proliferation plots are strange recently.
Please look at this picture: http://postimg.org/image/8e2pa9tg3/ or attached picture.
The upper row is my previous date that show standard graphs and samples.but recently my samples have high baseline fluorescent with a strange shape.
What is the reason for this problem?
My procedure briefly is:
I take liver RNA and then treat it with DNase to remove genomic DNA. Then I synthesise cDNA by Reverse Transcriptase Kit.
I use my cDNA to for real-time PCR by Cyber Green master mix. Standard is plasmid cloned.
Thank you
Masoud
There is too much cDNA/target in the reaction(s) showing the strange dark thick curves in your most recent runs. If my hunch is correct, those particular samples (that generate the early-appearing dark curve(s)) can be diluted ~1:1000 more than they are right now, then they should start adopting a good shape and will cross the Cq threshold more near the center of the picture. Right now, it appears you can't set the cut-off cycle low enough to accommodate the beginning of the dark curve amplifications...
Thank you, dear Jack, but how it can happen? Always I used 2ug RNA to make cDNA. I will do a run today with diluted samples to see how it goes. Thank you very much.
Masoud
You could have higher expression this time, or you used a new kit for making cDNAs which had a better efficiency of conversion to cDNA in those samples. Do you use an appropriate blanking buffer when you take spec or NanoDrop measurements to determine RNA concentrations? If dilution helps, then at least you know it is a concentration issue. I can't comment any further until the dilution test has been done.
Do you expect super-expression of this target from time to time - e.g., with treatment, etc.?
Thank you, Jack.
I used the same kit that I always use. It has a buffer5X, RTase, dNTP and random primer. I only changed the dNTP tube with a same new one. For blanking of nanodrop, I used water that is same with the water I use for resolving RNA. That Genes are Malaria 18s and mouse 18s as house housekeeping. Since I have this high baseline problem as you see in plots both Gene of interest and housekeeping (mouse 18s) this high baseline can be seen.
18S is notoriously abundant and often appears 15 to 18 cycles earlier than most other targets. So, knowing that you are experiencing this with 18S makes perfect sense. Those samples can be diluted 1:1000X more. But, it is curious that your gene of interest doing this same thing?: is your gene of interest hyper-abundant normally as well?
The Gene of interest is dependent to malaria burden in the liver. Usaually after normalization ratio is around 1.
Dear Jack
Your suggestion was amazing. I diluted my cDNA and it looks great. Although maybe 1000 times dilution is too much. I think I should do a titration to find a lower dilution factor. I attached 2 pictures of proliferation plots.
Thank you
Masoud
Looks hopeful! And, I agree, a titration study would reveal more. Good luck~!
May be your RNA degraded or you used a higher concentration of DNA.
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