Hi, I have been doing RNA isolation using the standard RNAeasy Plus mini kit. The 260/280 ratios seem fine - between 1.8 - 2-0, however the 260/230 ratios have been consistently low ( below 1.. as low as 0.3) or extremely high( above 2 or even 3) and I am unable to figure out why. I was using the same kit in my previous lab and following the protocol but I never had this issue. I do the extra spin step with an empty eppendorf to make sure that I have dried the column before elution with water.. I tried running a few of the samples on the Bioanalyzer to check the RIN values but the samples look fine..
I asked around in the department but either people were not facing this problem or they told me it did not matter so much .. so I am confused.. AND curious .. as to why this keeps happening with my samples.. what could i be doing wrong.. ? when I measure the RNA amounts on the nanodrop, I always keep the samples on ice and then I store them at -80deg C.
Would appreciate your suggestions / thoughts.
Thanks,
Zankruti
I was wondering if you ever found out what the reason is, similar problem, low 260/230 but good for tissue samples, just organoids that are low. Thanks!
I am currently facing the same problem with the Qiagen miRNeasy kit. Although some samples have adequate 260/230 ratios, most have very low purity and it seems to be just random. Nanodrop One shows that the problem is with Guanadine salts contamination, which are components of the wash buffers. I tried to dilute the buffers with absolute ethanol by double the instructed dilution and add an ethanol wash step before dry spin. Initially some samples had better purity but then others didn’t fare as well and again it seems very arbitrary. The only thing that can reasonably be to blame for this random poor purification is bad non-standardised spin columns that cause droplets of the wash buffers to be caught inside the column however long you centrifuge your samples and contaminate the RNA upon elution. I would opt for extraction by TRizol solely but it has its own shortcomings, too.
it may because of the lysis buffer. some lysis buffer has to be freshly or stored at 4C for up to a month.