If it is applicable, What should be the recommended concentration of exosoemms as the starting sample?
Hello Sawsan Mohammed Elhalawani
I use Qiagen miRNeasy Micro Kit (https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/mirna/mirneasy-kits/) for RNA extraction from EVs from different sources (including urine and cell-derived EVs). With this kit you will be able to collect both RNA and smaller RNAs (like microRNAs). The kit that you asked for it might be able to be used for EV RNA extraction but I believe that you will need higher amounts of starting material and you will not be able to extract small RNAs/microRNAs.
Then for cDNA synthesis I use 2 different kits:
1) for expression of large genes or abundantly expressed I use QScript cDNA SuperMix from QuantaBio (https://www.quantabio.com/product/qscript-cdna-supermix/)
2) for expression of microRNAs I use Mir-X™ miRNA FirstStrand Synthesis from Takara (https://www.takarabio.com/documents/User%20Manual/Mir/Mir-X%20miRNA%20First-Strand%20Synthesis%20and%20TB%20Green%20qRT-PCR%20User%20Manual.pdf)
Regarding the number of EVs to start the RNA extraction, you might need to do some testing but we use as minimum 1E10 total amount of EVs.
Good luck
Carlos Jesus thanks for your helpful answer, I have tried to isolate exosomal total RNA using Qiagen RNeasy Mini Ki, but the yielded RNA concentration is very low. I think I will consider ordering the miRNeasy Micro Kit. If I can ask one more question, should I start with a pellet of Exosomes or can I use exosomes sample suspended in about 100UL?
Sawsan Mohammed Elhalawani I would say that you can use the exosomes suspended in about 100 uL. The most important would be the amount of EVs that you have in that suspension. But in order not to interfere with the rest of solutions in the extraction kit, do not go further than 100 uL of your sample. Good luck!
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