I convert RNA isolated from MRSA to cDNA. Is it important to clean up that cDNA if I want to run a DNA microarray?
The concentration of cDNA by nanodrop around 2000 ng/ ul. After purification it became only 400 ng/ ul, using Genjet purification kit. What is the problem for that low concentration?
Instead of purification you can only dilute your cDNA about 10 fold. Providing you used enough RNA to start with, this dilution will decrease dramatically the influence of inhibitory molecules on your further cDNA analysis. Additional advantage is that you don't lose anything.
In a micorarray experiment It is important to load equivalent amounts of cDNA to every microarray to improve the quality of posterior data analysis.
After any RT reaction, you end-up with a mixture of template RNA (unless you degrade it), cDNA and free nucleotides that you load in excess to perform the RT rxn. All these components absorb at the same wavelenght and equally contribute to the measurement given by nanodrop. Thus, a cDNA purification step is recommended for a microarray experiment. And the drop in quantification by nanodrop is due to the clean-up of all these contaminants.
The only reason to not purify cDNA for a usual qPCR experiment is that it's usually too diluted to afford a good purification and quantification.
Just as a non relevant comment, In my experience, 400ng/ul after cDNA synthesis for microarrays looks as a good yield for (obviously considering my regular protocol).
thank you so much Dr. Silvia Rodriguez-Mulero .
I used QIAgene reverse transcription kit, which already degraded RNA. That kit contains Rnase H.
Good! You have a lot of free nucleotides then! (also from the template degradation)
Hi,
Effectively, a purification of the cDNA is important to remove contaminants notably those that could absorbe at 230 nm, and generate interferences in the hybridization process. By purifiying your cDNA you also improve your final signal/noise ratio.
I understand the concern of loosing sample during the purification but you should actually have enough cDNA.
Best
- Badges
- Science topic
- Similar topics
- Psychology
- Mental Processes
- Learning