I am using the Norgen Biotek Total RNA Purification Kit for isolation of RNA for RT-qPCR analysis. The RNA obtained is high-quality and in good quantity, but I have been having problems with RNA-binding columns breaking. Even when they don't break, some silica carbide is liberated during the elution step, and I am forced to spin down my RNA samples to pellet the silica, and then transfer the RNA to new, clean tubes.
Has anyone else had these problems with the Norgen columns or other columns? And do you know if even small amounts of silica can affect RNA quantification via UV spectroscopy, cDNA synthesis and/or qPCR analysis?
Thanks in advance!
I am using Quigen Kit for RNA isolation, I have never faced such problem. The only suggestion from me is, use other silica based binding columns, by using same kit.
And there is no significant effect of silica on RNA extracted.
Hope you will get positive results.
Cheers!!
Maybe your batch is defective. I used Qiagen Kit, now I use Mo Bio Kit and I don't have any problem with the binding columns. Use another columns with the same kit or change it. Qiagen works really good.
I wish you all the success.
Tània.
Contact the company or switch to a different kit system like Ambion, Qiagen or use TRizol.
I've certainly never had this problem, and I have used the Norgen kit quite a bit, so I would call the company. Provide them the lot number of your columns; it may be a bad batch. As for the effect of silica, there should be no effect on down stream applications. In the "old days" when we used glass milk, there was always silica left in the RNA. As for RNA quantification, it is a little more difficult to be certain... I'm wondering if the solid particles will block light, thus giving a false absorption. If quantification is critical and the amount of sample is plentiful, you could try filtering on a 0.22um membrane, but honestly I think you should be fine.
Hi Richard,
Below is a response from Norgen's team addressing your column leakage problem
"Thank you for your feedback. We always appreciate hearing any comments from our customers. Last year we have made a change to the manufacturing of our spin columns which now feature a new O-ring which will better secure the resin material within the column. We look forward to your continued feedback and helping further anytime"
RNA purification using Norgen’s kits is based on their proprietary resin, which is Silicon Carbide and not Silica, as the separation matrix without phenol. This give them the advantage of purifying all RNA irrespective of size or GC content, without bias unlike other silica-based column/phenol that is biased to bind specific sequences/sizes.
I experienced clogged Norgen columns in lysis step and also in wash step of RNA extraction. I also observed a bit of silica after wash in the bottom of tube.
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