RNA Degradation - DNAse I Treatment, 260/230 ratios (Feb/13/2009 )
I extracted RNA from cell culture samples for a microarray using the Qiagen RNeasy Plus Mini Kit, which uses a gDNA column for gDNA elimination. I also added DNAse I to the samples and incubated at 65C. After the 15 minute incubation, I realized that I forgot to add 25mM EDTA to the reaction. So I added the EDTA and re-incubated the RNA with DNAseI at 65C for 15 more minutes. After synthesizing cDNA, I read OD. The 260/280 ratio is low @ 1.5. The 260/230 ratio is very high in some samples (ranges from 2 to 6.5). The RT-PCR however seemed to work. I am very suspicious of the quality of this RNA and cDNA prep. Please advise....
Your RNA sample should not be used for microarray. The high 260/230 ratio indicates high salt concentration in your sample. For microarray, it is necessary to check RNA quality by running agarose gel or using bioanalyzer.
pcrman on Feb 14 2009, 01:19 AM said:
Sorry, I might be wrong, please correct me if so. Wouldn't we expect the A260/A230 Ratio be low if the salt concentration was high? I have been experiencing this prblem as well and I haven't been able to sort out what the hell is happening with similar A260/A230 Ratios (I've even got one at 20-something).
Try diluting your sample in 10mM Tris-Hcl, pH 7.4, not water. also low ratios might come from phenol contamination. High ratios might come from salts. I've never actually seen ratios as high as yours. But if you used Trizol, maybe try doing clean up using a column spin methods and try that.
asterix on Feb 13 2009, 08:43 PM said:
Had you checked your DNase I treated RNA on a gel? That would give you a visual confirmation (better than spec) if you have any RNA and DNA left. I don't understand why you need to do the DNase digest at 65C and why you would add EDTA to the DNase I reaction. DNase I works well at RT or 37*C and adding EDTA will remove the divalent ions required for DNase I activity. It makes no sense after doing DNase treatment without EDTA (correct way) and then you repeated it with EDTA (works poorly if any). Usually you add EDTA after the DNaseI reaction and then heat inactivate the DNase in the presence of EDTA to protect the RNA. I am puzzled why you would need to read OD of the cDNA reaction and there were tons of nucleotides (or maybe you had precipitated the cDNA?). It sounds you didn't know what you were doing buddie.