RNA not visible on gel :( - (Oct/13/2009 )
Hi
i did RNA isolation from mammalian cells and got pretty good yield (1ug/ul) with OD 260/280 values ranging from 1.8-2.0. I ran a 1.5% agarose gel and loaded 1 ug of RNA per well. I then stained d gel with EtBr for around an hour. i couldnt see any RNA band under UV....even though my marker bands were clearly visible. i thought the staining of the gel wasnt proper so i stained it overnight. still i coulnt see anything on the gel. i have rechecked the RNA quantity and quality (od 260/280) using 2 nanospecs. the readings are quite consistent. i tried loading upto 5 ug RNA also but in vain.
Please help me. My boss is gonna kill me as these samples are extremely important. i cant figure out the solution to this problem.
Thanks in advance for your suggestions.
DRN on Oct 13 2009, 11:44 PM said:
i did RNA isolation from mammalian cells and got pretty good yield (1ug/ul) with OD 260/280 values ranging from 1.8-2.0. I ran a 1.5% agarose gel and loaded 1 ug of RNA per well. I then stained d gel with EtBr for around an hour. i couldnt see any RNA band under UV....even though my marker bands were clearly visible. i thought the staining of the gel wasnt proper so i stained it overnight. still i coulnt see anything on the gel. i have rechecked the RNA quantity and quality (od 260/280) using 2 nanospecs. the readings are quite consistent. i tried loading upto 5 ug RNA also but in vain.
Please help me. My boss is gonna kill me as these samples are extremely important. i cant figure out the solution to this problem.
Thanks in advance for your suggestions.
are you sure you are using RNAse free reagents? are you running a formaldehyde agarose gel?
Never stain RNA gel after the run. RNA just gets digested. Either quick and dirty: run a stained EtBr gel (no longer than 30 min) or long and proper - find a protocol for a formaldehyde RNA gel., in RNAse free conditions. For quick and dirty you get an overall impression if the RNA is allright, just cloudy bands. With the proper method you'll see clearly.
Check first with not-important samples of course. Having a bulk of control RNA is always a good idea anyway.
2 microgram should be enough.
thanks for the reply....
i am sure i am using rnase free reagents. and since i am getting good od 260/280 ratios, i assume that my rna quality is good and is not degraded. also i thought that it might be a problem with my gel so i ran another agarose gel with RNA that was isolated a few months back and which i was able to see on the gel earlier as positive control along with my sample. this rna was visible on the gel but my sample rna was not 
. so i dont think theres a problem with my gel.
the only difference with my positive control rna and my sample rna is od 260/230 ratios which are very low (~0.5) for my samples.
but i am really not sure whether this would affect the visibility of rna on the gel or not.
i am still really confused about how even with a good od 260/280 ratio, i cannot see anything.