I want to extract microRNA (miR-10b) from BT-549 cell culture with miRNeasy Mini Kit - QIAGEN.
I was wondering what is the concentration to extract 6-10 μg of RNA?
The protocol is saying that the maximum is 1X10^6 cells.
Is 2X10^5 can work?
Thank you in advance!
Yes, this could also work, depending on transcriptional activity of your cell type. However, you have to avoid clogging of binding collums. Thus, it may be usefull to divide your smple after isolation and maybe after shredding to 2 binding collums.
1. At researchgate only, I noticed many question about low miRNA concentration by using miRNA isolation kit from amny company.
2. We are also working on miRNA, We prefer to isolate total RNA and then go miRNA-specific cDNA conversion (cDNA kit from Qiagen which converts all the miRNA present in the total RNA to cDNA) and then using miR primer (say for miR-10b), we go qRT-PCR.
3. Even the cDNA obtained after using Qiagen kit, we can go for qRT-PCR with SYBr green, no need of Taqman or any specific master mix.
---Total RNA benefit is you will never loose miRNA (in context of column size n all)
---No issue of master mix
I may not emphasised on the question you raise but i m just trying to put up a another scenario.
This is right. However, you will ever get a mix of mRNA (including pri-miRNA sequences, antisense sequences and other RNAs). These may interfere with measurement and the obtained results when trying to specifically analyse miRNAs. Thus, I would recommend to use the most adequate technique instead of the most cheap method.
Yes, u r also right. What I pointed, Total RNA method is also well documented and described technique. Moreover, these method is not-at-all cheap because Qiagen miRNA cDNA kit and same Qiagen miRNA specific primers comes at high cost, and the products maintains specificity.
We also used Thermo miRNA isolation kit where we faced similar issue of low concentration, then u tried total RNA method.
Regards,
Saurabh
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts