mirvana isolation kit - (Oct/26/2008 )
Dear all,
I am some questions about the mirVana miRNA Isolation kit. According to the protocol of the kit, there are isolation of total RNA, the enrichment of small RNAs and isolation of small RNAs from total RNA samples.
i) At this stage, we would like to perform taqman microRNA assay, so which method should I use to isolate RNA?
ii) Also, I would like to know under what conditions I will use the other two methods to isolate the samples?
iii) When will the enrichment of the small RNA be required? Thank you so much.
Samantha
I am some questions about the mirVana miRNA Isolation kit. According to the protocol of the kit, there are isolation of total RNA, the enrichment of small RNAs and isolation of small RNAs from total RNA samples.
i) At this stage, we would like to perform taqman microRNA assay, so which method should I use to isolate RNA?
ii) Also, I would like to know under what conditions I will use the other two methods to isolate the samples?
iii) When will the enrichment of the small RNA be required? Thank you so much.
Samantha
The answer to your question is NO. you don't need to enrich for small rnas, there's no need to do it for taqman assays and is not requested by the manufacturer. the enrichment would mean for u to use 2 columns instead of 1 and if u compare the results u'll see that it's not worth the cost of a column. i've been working with miRNAs for more than 3 years now but i can't think why u should enrich for small RNA.
In case u have doubts there's nothing like trying...
hope it helps.
Fizban
Thanks Fizban for your reply. Besides wasting a column, are there any other disadvantages for doing the enrichment, followed by taqman microrna array assay? I have read some journals in the the research groups have used Taqman microrna array assay. It seems most of them have used total RNA. However, I have sent an email to ABI and the staff replied me as follows. This really makes me pretty confused.
Samantha
Dear Samantha,
Regarding to the use of mirVana miRNA isolation kit for Taqman microRNA assay, procedure in the protocol of "enrichment of small RNAs" is recommanded after organic extraction for isoaltion of microRNA.
1. The protocol for " isolation of total RNA" is performed when total RNA is used for the experiment.
2. The protocol for "isolation of small RNAs from total RNA samples" is performed when you have total RNA in hand and want to extract the small RNAs from this batch of total RNA.
3. The protocol for "enrichment of small RNAs" is performed when small RNAs (e.g. microRNA) were extracted from the starting materials (e.g. cells, tissues).
The procedures in the protocol of " isolation of total RNA" and "enrichment of small RNAs" are performed after organic extraction.
The combination of "isolation of total RNA" and "isolation of small RNAs from total RNA samples" was performed when both total RNA and small RNAs are required for the experiments. Briefly, after isolation of total RNA, a portion of total RNA will be used to performed the isolation of small RNAs using steps in "isolation of small RNAs from total RNA samples" . Finally, both total RNA and small RNAs were obtained.
If you have any questions, please do not hesitate to contact me.
Best Regards,
Amy Lam
Application Support Specialist
Applied Biosystems Hong Kong Limited
Tel: 852 3107 7623
Mobile: 852 6478 8161
Fax: 852 2756 6968
Email: Amy.Lam@appliedbiosystems.com
Samantha
Dear Samantha,
Regarding to the use of mirVana miRNA isolation kit for Taqman microRNA assay, procedure in the protocol of "enrichment of small RNAs" is recommanded after organic extraction for isoaltion of microRNA.
1. The protocol for " isolation of total RNA" is performed when total RNA is used for the experiment.
2. The protocol for "isolation of small RNAs from total RNA samples" is performed when you have total RNA in hand and want to extract the small RNAs from this batch of total RNA.
3. The protocol for "enrichment of small RNAs" is performed when small RNAs (e.g. microRNA) were extracted from the starting materials (e.g. cells, tissues).
The procedures in the protocol of " isolation of total RNA" and "enrichment of small RNAs" are performed after organic extraction.
The combination of "isolation of total RNA" and "isolation of small RNAs from total RNA samples" was performed when both total RNA and small RNAs are required for the experiments. Briefly, after isolation of total RNA, a portion of total RNA will be used to performed the isolation of small RNAs using steps in "isolation of small RNAs from total RNA samples" . Finally, both total RNA and small RNAs were obtained.
If you have any questions, please do not hesitate to contact me.
Best Regards,
Amy Lam
Application Support Specialist
Applied Biosystems Hong Kong Limited
Tel: 852 3107 7623
Mobile: 852 6478 8161
Fax: 852 2756 6968
Email: Amy.Lam@appliedbiosystems.com
the only thing u can do is compare miRNA expression in 1-2 samples in which u do both extractions. i did it and found that total RNA isolation is suitable for my kind of investigation. when i don't use miRVANA i use total RNA extrcted with trizol. no need for enrichment for me, it depends on your needs. In my opinion total RNA is just fine.
bye
Fizban
I know ABI and Ambion are now the same company, but I've found the Ambion staff to be more knowledgeable about their products compared to ABI. The email that you posted looks like a generic email that they have and not something specific for your question. However, I agree with Fizban, there is no need to enrich for the small RNAs. You only need a small amount of total RNA for the qRT-PCR anyway, I don't think there is any need to enrich.
Just got a reply from the Ambion Technical Support. They say total RNA should be used for Taqman miRNA microarray assay.
Thanks Fizban and miRNA_man.
Samantha