I am using RNeasy plus mini kit for extraction of RNA from E. coli. I am following the procedure as written but when I run my eluted sample on the agarose gel to see the RNA integrity (which should show the 16s and 23s RNA bands), I don't see any bands, only the ladder. But when I use the nanodrop, it shows me that there is an RNA with a concentration not less than 3ng/µl. can someone tell me why there are no bands and what does this reading means?
Thanks
I think you need more RNA, i had read in protocols that for view the two bands in agarose gel is required 1ug of RNA. How much bacteria do you take up for RNA extraction?. Another point is, i dont know if you are doing a denaturing agarose gel, using formamide, formaldehyde and MOPS buffer, because otherwise, will be very hard view the two bands, 28S and 18S.
I think you need more RNA, i had read in protocols that for view the two bands in agarose gel is required 1ug of RNA. How much bacteria do you take up for RNA extraction?. Another point is, i dont know if you are doing a denaturing agarose gel, using formamide, formaldehyde and MOPS buffer, because otherwise, will be very hard view the two bands, 28S and 18S.
Hi Sinan Baho, We also had this problems in our lab. the Nanodrop is not the best way to quantify RNA, it also detect unintact RNA. We now use the Zymo mini column and the yield that we obtain is great comparing to other columns such as the Qiagen mini
I think you should load at least 400ng RNA, otherwise you will probably see nothing (or very weak bands) because this method is not that sensitive. Regarding the denaturing agarose gel, we use a "normal" 2% gel in TAE and have no problem seeing the 2 bands.
I usually don't trust Nanodrop measurements below 10 ng/ul based on my previous experience. It's mostly just noise. If you have such a low concentration of RNA I recommend using Qubit or something else that is more sensitive - perhaps Bioanalyzer or Tapestation depending on what you have access to.
How long do you run the gel.. and which buffer you use? try to load atleast 0.8-1ug RNA in 1.5-2% gel, using A denaturing buffer (MOPS is best) .. do it at low voltage and there you are.. hopefully you will see the bands if you still have RNA in the samples... (depending on number of cells used for extraction).. I believe that you are diluting your buffers in DEPC water .. good luck
Dears All..thank you very much for your answers. Dears Ibsan Valerio and Ayesha Maqbool, I am using normal gel without a denaturing buffer (MOPS) but I think the problem is in the sample loaded, I am using less than 109 cells in 100microliter. Can anyone give me a better way for extraction from a biofilm?
Thanks
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