I want to isolate exosomal miRNA only, for that do I need to first isolate total RNA and then go for miRNA isolation? or can I directly isolate miRNA from exosome and do the analysis? if yes please recommend the kit for this protocol.
Hello Himadri Pat,
I hope you find the following articles useful.
Article Isolation and Identification of miRNAs in exosomes derived f...
Article Comparison of isolation methods of exosomes and exosomal RNA...
Dear Himadri!
You also look at this protocol, please:
https://www.nature.com/articles/s41598-018-28748-5
miRNA isolation and quantification
Exosomes obtained from all methodologies were similarly processed. All the processes were carried out in an RNase-free area, using RNase-free tubes and following the necessary measures to avoid miRNA degradation. Total exosomal RNA extraction was carried out using miRCURY RNA Exosome Isolation Kit (Exiqon) following the manufacturer’s recommendations. First, 1 µL of spike-in mix (UniSp2, UniSp4 and UniSp5) was added to 60 µL of lysis solution, mixed with 200 µL of exosome solution and incubated at RT for 3 min. Then, 20 µL of protein precipitation buffer was added, vortexed and incubated for 1 min at RT. Samples were centrifuged for 3 min at 11,000 g before collecting the supernatant and adding 400 µL of isopropanol. This solution was loaded onto the microRNA Mini Spin Column BF columns. Different cycles of washing were performed until the final elution of RNA. To recover total RNA, we used 100 µL RNase-free H2O. Total RNA was stored at −70 °C. The miRCURY LNA Universal RT microRNA PCR kit (Exiqon) was used for reverse transcription. Before the RT reaction, 0.5 µL of another spike-in mix (UniSp6 and cel-miR-39) was added. cDNA was stored at −20 °C until use. Just before using, cDNA was diluted 1:80 in nuclease-free H2O. Finally, RT-qPCR was performed using 4 µL of diluted cDNA, 5 µL of PCR Master Mix (Exiqon) and 1 µL of LNA PCR Primer Mix. In addition to the primer set analyzed, 4 primers corresponding to UniSp2, UniSp4, UniSp5 and cel-miR-39 were used to assess sample integrity during RNA extraction, RT and RT-qPCR. The PCR reaction was run in an Applied Biosystems ViiA 7 system using the recommended manufactured protocol.
I would first isolate the exosomes from the cell. Then do a regular total RNA extraction that also include the small RNAs. The miRVana kit has worked very well in our lab.
if you dont separate exosomes from the rest of the cell its no way to know which miRNAs that are from exosomes and which ones that are cytosolic. The extraction of totalRNA together with small RNA have never interferred with our downstream analysis of miRNAs and has the benefit that you can check quality by measurements of the common absorbtion ratios. Hope this helps!
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