Dear all, I have extracted RNA from urine samples using the Phenol-Chloroform extraction method and obtained the yield (~83ng/uL) on Quantifluor Fluorometer. But I could not get a clear band on the gel. I am attaching the gel picture (first two wells have been loaded). Could you please tell me what is wrong here? As I am a new one for RNA work, I need help from you to improve my technique.
Dear Sumana Sivalingam,
The two dark lines on your gel are caused by the loading dye that you are using, you can fix that by reducing the concentration of the dye.
Unfortunately I don't see the two distinctive 28S and 18S RNA bands, but it looks like you have genomic DNA in your wells (DNA usually runs much slower through the gel than RNA). Did you treat your samples with DNAse?
One of the problems might be the presence of RNAses, but I guess that you took care and used nuclease free plasticware and you cleaned properly your equipment and gel trays/combs before using them.
Which kit did you use for the Quantifluor Fluorometer? As far as I know the the QF RNA System and Quant-iT RiboGreen RNA Reagent are not specific for RNA and will bind to DNA as well which means that the presence of DNA in your RNA sample might lead to overestimation of the RNA concentration. Only Quant-iT™ RNA Assay uses RNA-specific dye.
Best,
Valentina
Thank you valentina for your reply. Yeah. I extracted again RNA and got RNA like one band. I need to trouble shoot that why I got only one band? Could you please help me?
Hello Sumana Sivalingam,
If you can share a photo of your gel along with some additional information we will be able to provide some advice with more confidence. Are you using denaturing or native agarose gel? What was the density of your gel? How much RNA you loaded and with what dye?
My wild guess is that either your gel density is too high (0.8 or 1% should be enough and it provides good resolution) or that your RNA is partially degraded and you can't see the 28S RNA band. Did you see any smearing on your gel?
It's a good idea to load some size marker as well - that might help you to determine the size of the bands and it will serve as a control that your gel ran properly.
In your previous post you mentioned that you measured the yield with Quantifluor Fluorometer. What are the new concentrations? Did you measure the A260/280 and A 260/230 ratio with some type of spectrophotometer?
Best,
Valentina
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