I ran a RNA agarose gel before doing the RT expecting to see two bands 28S and 18S rRNA subunits but I see an extra band above the expected ones. It does not seem to be gDNA...
The samples come from N2a cell line transfected with LV miRNA extracted with Trizol.
Could this be residual vector?
Not all of the vector DNA may have partitioned into the interphase/organic phase and/or a portion of the interphase may have been removed with the aqueous phase after the initial separation.
I think Sabine has made a really good suggestion - I can't think of anything else it COULD be that would produce such a sharp band, as gDNA contamination would look like a high MW smear. What's the sample on the far right of the gel - is that a non-transfected control? If it is, it would add even more weight to the 'residual vector' case. Another way you could possibly test this is to DNase treat the RNA - give it a long incubation given that if it is DNA, it's a lot of DNA! If the band goes away or at least reduces in intensity by a large amount, that would be very suggestive that it's plasmid. There are some differences in how plasmid partitions in an extraction/separation vs gDNA, which is the basis for our ability to separate plasmid from gDNA in mini/maxi kits etc.
The first column (the left one) is a non transfected RNA extraction control, the sample on the far right of the gel is human RNA extraction, a lab partner asked me if he could use one of the empty wells (I should had to delete that from the image before I uploaded it).
I dont't think its gDNA, I preferred to leave volume to pipet near the interfase
We (Laura Leighton and myself) suggested that the RNAs of the transfected samples are "contaminated" withe vector/plasmid DNA.
see troubleshooting guide
https://www.thermofisher.com/at/en/home/technical-resources/technical-reference-library/nucleic-acid-purification-analysis-support-center/rna-sample-collection-protection-isolation-support/rna-sample-collection-protection-isolation-support-troubleshooting.html
If the DNA contamination was detected in RNA isolated from cells after transfection with a plasmid, not all of the plasmid DNA may have partitioned into the interphase/organic phase once the chloroform was added to the TRIzol® Reagent. If RT-PCR is being used to assay gene expression from the transfected plasmid, a DNase I treatment will be needed.
Only, in the first lane (far left) in the RNA extracted from non-transfected cells there might be some residual genomic DNA, indicating that there is room for improvement in the RNA isolation procedure, while the RNA on the far right of the gel looks very good and free of genomic DNA.
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