Hello,
Today i want to ask about stability of cDNA.
As far as i know, cDNAs are very stable molecules compared to RNAs.
During RNA works, I use my personal tools(Pipet tips, 1.5ml tubes, etc)
to minimize contamination considering low stability of RNAs.
In contrast, After cDNAs are synthesized during RT-PCRs,
I just use tools
others are also sharing, not using my personal tools
because I think cDNAs are very stable.
Should I also use my relatively clean, personal tools above
when working with cDNAs?
I want to make clear about my question : I never use
tips and tubes others already used.
I use my tools in containers only used by me in RNA works
but I just use remained tools in containers others already opened for
their works during cDNA works.
I hope everyone do not misunderstand what I exactly mean
Thank you!
cDNA is stable as it's already DNA. If your next step is qPCR, them you should follow the recommendations to avoid PCR amplification carryover contamination that include more tubes and tips.
Lee,
It is a matter of presence of contaminating nuclease activity in your solution. If your solutions are nuclease free and your tools as well as technique ensures that no contamination takes place post first strand cDNA synthesis, the product will be quite stable when stored frozen.
The quality of reagents an technique cannot be overemphasized when it comes to working with mRNA. Clean mRNA dissolve in TE remains substantially intact at room temperature for days. If one sees inadequate amount of cDNA, its often because the quality of RNA was poor to begin with, not that the cDNA is inherently unstable.
Dear Nina Odermatt,
Thank you for your great and informative answer!
I learned a lot thanks to your help!
Dear Julio Raul Fernandez Masso,
Thank you for your helpful and quick response!
It was great pleasure for me.
Thanks again.
Dear Tausif Alam,
Thank you for your helpful answer!
so If i get you right, you mean that when it comes to
using tools in cDNA works(cDNAs from RNAs),
there are no possible problems other than risks of nucleases which degrade my cDNAs?
I appreciate your help again!
One you understand the sources of nuclease contamination and precautions needed to avoid them, it will be much smoother sailing.
I am not necessarily recommending that it is fine to store mRNA at room temperature (it wasn't by design, one of my students forgot tubes at room temp) but it does illustrate the point that if your final products are nuclease free, the DNA (and even RNA) is quite stable.
If you suspect that shared pipettes may be contaminated, either decontaminate them or don't use them.
Thank you so much Tausif Alam,
Sorry for additional asking, but English is not my first language so I occasionally experience difficulty in communication by English in Researchgate.
Yes, preventing nucleases is very important point in our conversations, but I want to ask whether there are other
possible problems other than nucleases when it comes to
using tools when working with cDNAs synthesized.