low OD260/280 ratio for RNA extraction with TRIzol - (Jan/12/2007 )
Hi everyone, i am puzzled with this problem. I tried twice with TRIzol for my RNA extraction from cells cultured in 35mm dishes, the OD260/280 ratio is 1.6 and 1.5 respectively. I just follow the protocols from invitrogen.
the only difference is when i did spectrometry for quantification, i used a cuvette used for DNA quatification. Will that lead to the low ratio?
I haven't run RNA gels to see the quality of RNA. Do you think i still need to run a gel?
Thanks a lot!
You can use same quartz cuvettes used for DNA but it should be washed and dried prior. I do not buy the results given by spec completely even after cross checking with more than one instruments. Even though running on gel will not give you any idea about 260/280 or 260/230 ratio still its best to check RNA integrity on gel. After confirming two crisp bands and no degradation I go ahead for further work. Although recommended 260/280 and 260/230 values are >=2 and >=1.8 a little less ie ~1.7 have worked fine with me.
Thanks a lot!
I am going to run the gel. Do you think a native gel is fine or shall i run a denatured gel?
Recommended is running the RNA sample on denaturing gel(formaldehyde gel and MOPS buffer). What I do is when I have multiple RNA samples at a time I run multiple samples with a cotrol quantified sample. But if you are sure of providing RNase free condition while running gel(eg sds wash of tank, using rnase free water ,dye and buffer) you can use normal agarose gel.
i use normal agarose gel.. senior told me that agarose gel enable to detect genomic DNA, can know whether got DNA contamination or not...
i didnt face wif serious RNA degradation problem by using agarose gel.
I routinely used non-denaturing agarose gels to check my RNA following the TRIReagent protocol and it worked fine with me. As Parami said, one can also spot gDNA.
In addtion, attention should be payed to the RNA pattern on the gel. Three sharp bands should appear, and it is important to have a nice top (i.e. heaviest) band. If that one is faint, you can seriously consider to trash everything straight away.