Hi researchers,
I have to perform an RNA extraction from isolated exosomes of serum samples. I tired trizol. However, the concentration of RNA is so low and I can not perform RT-PCR analysis for gene expressions.
Does anyone know what is the best commercial kit for RNA isolation from exosomes of serum samples? I previously isolated exosomes by ultracentifugation. Now, I purchased miRCURY Exosome Kit and try new exosome isolation.
Is there any suggestions?
Thanks
hi
Article Optimizing exosomal RNA isolation for RNA-Seq analyses of ar...
good luck
Hello Gamze
Exosomes carry low level of RNA. Exosomes from 4 ml of serum yields approximately 1-10 ng of RNA. The amount of RNA will also vary among individuals depending on many factors like the age, diet and health status. TRIzol or TRIzol LS usually gives higher yield of RNA that any commercial kit. I always use TRIzol method for RNA isolation. But purity is compromised. You can use the RNeasy mini kit from Qiagen. Reza has provided a good reference. You can follow that too.
Thank you.
Dear Gamze Guney Eskiler,
I agree with Malcolm Nobre that TRIzol or TRIzol LS provides better yield. But as he rightly mentioned - purity is a big concern. Recently, we adopted a two-step approach and the results have been satisfactory.
1st step: We used Thermo's Total Exosome Isolation Kit (cat. No. 4484450) and were able to get reasonable quantum of exosomes from plasma . We characterized the isolated population using anti-CD9, anti-CD63, and anti-CD81 antibodies.
2nd step: For isolation of RNA and protein from the enriched exosome population (from 1st step), we used Total Exosome RNA and Protein Isolation Kit (Thermo cat. No. 4478545).
Hope this helps !
Best - Pradyumna
Pradyumna Kumar Mishra thank you for your response . I order thermo total exosome isolation kit. Your recommendation is very helpful for me
Reza Hadavi I read this article. I can not select which one is the best for RNA isolation after using miRCURY Exosome Kit. Thus, I determine two kit RNeasy mini kit and Thermo total isolation kit. Thank you for advice.
Malcolm Nobre , I tried Trizol. However, I can not obtain any results from RT-PCR even the expression of actin. Therefore, I choose commercial kit exosome isolation rather than UC. Is cDNA synthesis kit affect the RT-PCR result? I use High capacity cDNA isolation kit.
Hello Gamze
The RNA should be of good quality. If you have a bioanalyzer in your lab check for RNA integrity. As you know total RNA extracts usually contains mRNA, t RNA,
r RNA subunits and small RNAs. For gene expression and transcriptome analysis mRNA is used. mRNA is converted to cDNA by reverse transcriptase enzyme which catalyzes cDNA synthesis starting with the polyA tail on mRNA. So the RNA extracted must be of good quality giving you intact mRNA. c DNA kit will not affect your RT-PCR results if your template RNA is of good quality. Then you can go ahead with your experiments. You are on the right track. UC will not give as good results as the commercial kit.
All the best.
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