Hi Khonaw,

No, it is not necessary to do further clean up. This is a column based kit and the RNA should be clean (however some residual DNA contamination cannot total excluded).

After elution of the RNA you should use a photometer to maesure the concentration and get information about the purity of your isolated nucleic acid.

You should also do agarose gel gel electrophoresis to see if the RNA you isolated is intact. For further information check the handbook. You can download this from the Qiagen website (if you don't have it already): https://www.qiagen.com/de/resources/resourcedetail?id=5ea61358-614f-4b25-b4a5-a6a715f9d3aa&lang=en

Hi Kesper

Thank you for your reply. I had downloaded the handbook for the kit. Within the handbook, there is an RNA clean-up protocol. Do I have to follow this protocol?

Do I use Nanodrop instead of gel agarose gel electrophoresis?

Does the DNA contamination affect on investigation detection of m6RNA and mapping?

Hi Khonaw,

the RNA clean-up protocol is a protocol for cleaning up RNA after other applications like enzymatic reactions and/or DNAse treatment.

However, if you follow the normal protocol for RNA isolation from blood, tissue or cells without the separate DNAse digestion (Appendix E in the handbook) you don't need to do this step.

The DNase digestion is recommended if you have a downstream application which is really sensitive to DNA contamination like real-time PCR for low abundant RNAs. I assume with m6RNA detection and mapping you are refering to the analysis of m6RNA methylation?

As I have never done this I am not sure if genomic DNA contamination might be a problem, however I would always try to avoid it as much as possible.

One tip which might help to avoid DNA contamination is to not overload the column. This means do not exceed the maximal amount of blood, tissue or cells recommended in the protocol (if you overload the column it is not only more likely to have genomic DNA contamination it might also decrease your ovall amount of isolated RNA).

Regarding your question if you can use the Nanodrop instead of the gel electrophoresis you should do both.

The Nanodrop will give you information about the concentration and the purity of your RNA (it will not show DNA contamination) but you will not get information about your RNA integrity.

As RNA is especially prone to degradation you should always check your RNA integrity on a gel. In this article you find a good picture how intact total RNA should look like on a gel: Article The effect of Sclareol on the expression of MDR-1 gene and G...

Please be aware that with this kit you will not clean-up RNA with a size below 200 bp.

I hope this helps