How to explain the fact of having two bands in electrophoretic profile of cDNA simples? Knowing that RNA extraction was made on a plant tissue, with control individuals and others subjected to abiotic stress. the cDNA synthesis was done using oligodt.
There are many reasons. What do your controls look like? The most obvious reason is that you also amplified genomic DNA along with your cDNA.
The controls are individuals on whom no stress has been exerted. The intensity of the two bans on the gel were similar for each extract.
Do you think that I need to add more DNase before cDNA synthesis ?
Dear Fella, such appearance is due to the use of random hexamers and / or oligo DT primes present in the cDNA (RT) kit. Whatever cDNA is generated from such primers, it will be definitely of variable sizes, especially those generated by random hexamers, which bind to various spots at your RNA and thus generate various-length products, which are not heavy enough to give you distinct bands, unlike in genomic DNA case. So often smeary or dimmed multiple bands appear for the cDNA on gels. So, it is a normal phenomenon and no need to get much worry for it.
That is why it is highly recommended not to check the quality of cDNA on gel and never check cDNA quantity through a Nanodrop or any other UV-based equipment, these both are wrong and useless approaches, whereas you can save your lots of time and resources by not doing this tedious protocols. Simply check your cDNA quality as undiluted and as well in few proper dilutions set by running any good housekeeping or reference gene through conventional PCR or by qPCR...
Regards.....
How big are the bands? If one is the expected size for cDNA and the other is the expected size for gDNA, then you have genomic contamination. If the unexpected band is about 100 bp or smaller then it's probably a primer band. What does your negative control with just water look like?
Your controls should also include: water, genomic DNA, no RT.
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