Dear Viral, in terms of RNA you must get three distinct bands for 23,16 and 5S in an agarose gel. I am not much sure about the technique you are using, but presence of 5S is perfectly alright. 

From the position of the small peak close to the 5S, I would say that you are detecting the tRNAs, so nothing to worry about. You can run the sample on a PAA gel next to an appropriate ladder for confirmation, the bands should migrate between 70-90bp. 

I would rather be concerned about the great imbalance of extracted 5S and 16/23S. Is the kit/protocol you used designed for specific isolation of small RNA?

I fully agree with Tanino. The peak of 5S RNA should be much smaller compared to the 23S and 16S peaks. It is highly likely that you co-purified the tRNAs along with the ribosomals and messengers. Indeed easily checked on denaturing PAA gel

The fact that the concentrations obtained with the Nanodrop and Bioanalyzer differ is due to the different way of measuring (Nanodrop = UV absorbance and Bioanalyzer is Fluorescence intensity). I always stick to the Bioanalyzer concentrations because the intensity is directly related to the amount of dye bound to DNA and for the Nanodrop you will include other components that absorb at 260nm. But probably there is a deviation in these values as well.

Hi Tanino, Thank you for your input. I am not sure why is there a great imbalance for 5S and 16/23S. The showed darker 5S band than the other two. It is difficult to explain whether these samples are good enough for RNA-seq. 

Dear Frank, Thank you for your explanation.

Do you guys think if big peak is due to using DEPC water for elution? I used RNeasy kit (Qiagen).