I have recently extracted total RNA from the bark of Rubber tree. However, I am not sure if these RNA's are contaminated with gDNA? I see some smears on the RNA. Could the smears represent gDNA?
Large quantities of genomic DNA (unsheared) will be of such ridiculously high molecular weight it will remain in the loading wells, so you're ok there. Large quantities of sheared DNA will produce a smear effect, but...so will total RNA. And small quantities of either variety of gDNA will probably not be detected by this method.
For simple gel electrophoresis you really just look for two relatively sharp ribosomal bands, which you have. If your RNA was degraded it would be just a vague low mol wt smudge at the bottom.
So: you have fairly decent looking RNA, which may or may not be contaminated with gDNA.
Questions: is it important to be sure you have no genomic DNA? If so, a simple treatment with DNAse will get rid of DNA content (just be sure to get rid of the DNAse before moving to cDNA synthesis).
If you're planning to use this RNA for cDNA synthesis and qPCR, design primers to span large introns: this way even in the presence of relatively high gDNA contamination you will only amplify cDNA template.
Large quantities of genomic DNA (unsheared) will be of such ridiculously high molecular weight it will remain in the loading wells, so you're ok there. Large quantities of sheared DNA will produce a smear effect, but...so will total RNA. And small quantities of either variety of gDNA will probably not be detected by this method.
For simple gel electrophoresis you really just look for two relatively sharp ribosomal bands, which you have. If your RNA was degraded it would be just a vague low mol wt smudge at the bottom.
So: you have fairly decent looking RNA, which may or may not be contaminated with gDNA.
Questions: is it important to be sure you have no genomic DNA? If so, a simple treatment with DNAse will get rid of DNA content (just be sure to get rid of the DNAse before moving to cDNA synthesis).
If you're planning to use this RNA for cDNA synthesis and qPCR, design primers to span large introns: this way even in the presence of relatively high gDNA contamination you will only amplify cDNA template.
If you plan on using for RNA-Seq then you definitely need to DNAse. I like Ambion's turbo DNAse kit b/c it uses a deactivating reagent (citric acid) rather than a heat kill step (I just hate exposing RNA to ANY amount of heat if I can help it).
I had the same experience as you are. Rubber tree has a lot of properties in their bark, especially mucus, latex and carbohydrates. Visually you have a good quantity of RNA. The smear you see normally is a disrupted RNA which happens mechanically during your extraction. Would you please tell me, what kind of method you use for extraction? Ultracentrifugation? Cesium chloride cushion? Try to use DNase to make sure your exact quantity of your RNA and run on the gel after. Scott's suggestion is good.
I should have added that the two ribosomal band look pretty clean. Doesn't appear to be a lot of degradation. If possible, running on a Bioanalyzer would give you a useful RNA integrity number (RIN) and a better idea of the quality of your RNA.
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