I used expired (1 year) DNase (along MgCl2 during RNA purification and got abnormal 260/230 as well as very low concentration. Before purification the conc. was high (180ng/uL), however after DNase treatment (with MgCl2) it became 7.8 ng/uL. The 260/230 became 3.5 and 4.5 (two samples).
HI Asiful,
Depending on what is the context of your RNA isolation, I would recommend not to use a DNase treatment. In my experience, I consistently see RNA degradation after DNase treatment and I only use this approach when keeping RNA integrity is not necessary. If you need to get really pure RNA, I would recommend to do an extraction with acid-phenol-chloroform (which will reduce the amount of DNA that you carry over from your extraction) and design a PCR set of primers that would allow you to detect if you have DNA contamination in your sample.
Good luck!
Anna
The true question is: your expired DNAse I still work? In my experience, usually if enzymes are stored properly and handled properly they're functional also after expiry date. I used sometimes in the past restrictions enzymes out of 2-3 years from expiry date but they were functional. The thing you only should take care about is eventual RNAse contaminations of old DNAse aliquots. Old reagents are handled and used more than once, maybe by different researchers, this increase the risk of contaminations. You should avoid contaminations of this kind of reagents more than every other thing..
About RNA purification after DNAse digestion, what kind of protocol did you used? If you're using acid-phenol/chloroform phase separation + isopropanol RNA precipitation you should check if all your reagents are free of RNAses contaminations. The loss of a small amount of RNA after purification is absolutely normal, you'll leave a small bubble of water during the two collection step of the upper phase. but not at this level anyway, something gone wrong probably.
And also the abnormal 260/230 ratio. You should ensure to have no phenol contaminations and carryover, along with RNAses contaminations, this may give you abnormal ratios and low yield.
Hi Md Islam,
Although kits may work well past their USE BY dates without a 'noticeable' problem, you will be working outside the time window the manufacturer can guarantee its optimum performance. This would mean you are not quite sure of how repeatable your experimental results would be.
That said, however I would think through what the RNA is intended for before deciding whether to go ahead or not. This would determine the kind of losses or aberrations you may accept. Also expiry may in fact relate to a single (or few) product(s) in the kit.
The other issue relates to handling of the products generally. The 'expiry date' is of course tied to optimum storage and handling of products and sometimes is at best a guideline, making part of a package.
While I do understand that economic realities are tough these days, it might be interesting to consider how much you would be planning to reveal when reporting these data. Would you let your reader know that you used an expired stock, or would you 'overlook' this small piece of detail. If you did overlook it, would that still be in line with accurate reporting? Would you (without prior testing) declare the kit usable a year after suggested expiry date? But if you did report that the kit was expired, what sort of information would you be required to give?
Small issue, many considerations, hard economic times!
Good luck.
Aman
Expired enzymes are usually functional if they have been stored properly with minimized thawing/freezing cycles.
You don't necessarily need DNAse treatment for RNA extraction. design proper primers at the intron-exon junctions.
run a agarose gel to check integrity of your sample.
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