I am isolating Exosomal miRNA from the blood plasma of buffalo. But miRNA concentration always varies from sample to sample. Sometimes I get high as 40ng/microL but sometimes as low as 3ng/microL from different samples using the same starting volume and same protocol. why is it so? I am using miRCury Exosome isolation kit Serum/plasma and Qiagen miRNeasy advanced kit.
Try extracting miRNAs 7 times from the same sample and see what is you reproducibility level. If it varies more then 10% there is a problem with the method.
what method you use to check miR conc.? nanodrop is quite unreliable in this, however fragment analyser can give you more dependable results.
you can use spike in controls during your extraction to see if there is any problem with your protocol. if the extraction is OK, then proceed with your experiment as any difference in the starting conc of miR, will be accounted for during the normalization step.
Rana Mumtaz Raoof Thanks for your kind response. I am using Nanodrop to check miR conc. I use miRcury exosome isolation kit followed by MiReasy advanced plasma kit.
Yes, I also think that any difference in the starting conc of miR, will be accounted for during the normalization step.
exosomes contain extremely small amount of RNA cargo (miRNA and other), so this input is always on the very "low end" of RNA isolation kits. besides that miRNA levels and content vary donor-to-donor, and even within the same donor- during the day, and depending on the diet and other factors. And obviously kits are "not all equal", you can try the Total exosome rna/protein isolation kit- based on parental mirVana paris platform (Invitrogen)
Based on donor condition and parental cell, exosomes could vary, we can't expect they will have same amount of miRs, cytokine and protein as they form randomly in a cell.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA