I am working on a project to look for valuable miRNAs for cancer diagnosis. I need to get the miRNA from exosomes.
Has anyone ever done this before and how did you do that? Thank you!
Hello Juefei,
There are few variables to consider when it comes to miRNA analysis. First, the EV purification method. Different methods with isolates slightly different species, thus miRNA profile may be different. Ideally you want as clean preparation as possible without contaminations with non-EV vesicles. Second, to have a good RNA isolation technique. Not just the kit, but practice, to avoid RNA degradation. Choose a kit that isolates small RNAs, since this is what you are interested in. Norgen Biotech has a nice kit for exosomal RNA isolation. And do RNA QC on every step to make sure there is no degradation.
Good Luck!
The quantity and quality of RNA depend on the purification methods of EVs. I personally used differential centrifugation with the SEC. you can find the EV purification protocol here 10.3390/ijms21155365 and the RNA extraction protocol given below
Required material
i. TriZol reagent
ii. Chloroform
iii. Glycogen
iv. Isopropanol
v. Ethanol
vi. RNAse free water
vii. Bioanalyzer chips and reagents (High sensitivity RNA kit)
RNA extraction protocol
i. Add 0.7 ml of TriZol reagent to concentrated EV sample (preferably > 1x 10 11 EV
per ml).
ii. Homogenize by pipetting.
iii. Incubate the homogenized sample for 5 minutes at room temperature
to permit the complete dissociation of nucleoprotein complexes. Centrifuge to
remove cell debris. Transfer the supernatant to a new tube.
PHASE SEPARATION:
Add 0.2 ml of chloroform per 1 ml of TRIZOL Reagent. Cap sample tubes
securely. Vortex samples vigorously for 15 seconds and incubates them at room
temperature for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 x
g for 15 minutes at 2 to 8 0 C. Following centrifugation, the mixture separates into
a lower red, phenol-chloroform phase, interphase, and a colorless upper
aqueous phase. RNA remains exclusively in the aqueous phase. Transfer the upper aqueous phase carefully without disturbing the interphase into a fresh tube.
Measure the volume of the aqueous phase (The volume of the aqueous phase is
about 60% of the volume of TRIZOL Reagent used for homogenization).
RNA PRECIPITATION:
Precipitate the RNA from the aqueous phase by mixing with isopropyl
alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIZOL Reagent used for the
initial homogenization. Add 10 µg of glycogen to the solution. Incubate samples at
RT for 10 minutes and centrifuge at maximum speed for 30 min at 4 o C. The RNA
the precipitate, often invisible before centrifugation, forms a gel-like pellet on the side
and bottom of the tube.
vi. RNA WASH:
Remove the supernatant completely. Wash the RNA pellet once with 75%
ethanol, adding at least 1 ml of 75% ethanol per 1 ml of TRIZOL Reagent used for
the initial homogenization. Mix the samples by vortexing and centrifuge at max
speed for 1 minute at 4 o C. Repeat above washing procedure twice. Remove all
leftover ethanol.
vii. REDISSOLVING RNA:
Air-dry or vacuum dry RNA pellet for 5-10 minutes. Do not dry the RNA
pellet by centrifuge under vacuum. Dissolve RNA in 20 µl of DEPC-treated water
bypassing solution a few times through a pipette tip. Heat the RNA solution for 10
min at 60 o C.
viii. SPECTROPHOTOMETRIC ANALYSIS:
Use the nanodrop to measure the concentration and purity of RNA (standard protocol
attached).
Use the Bioanalyzer to measure RNA quality
Total exosome RNA and protein isolation kit - it is based on parental mirVana platform, doing great job at recovery of small RNA esp microRNA. We used it for many studies and publications
Good results will also depend on the method of collection - if using blood then what are you doing to take serum or plasma and what volume? if plasma then which anti-clotting agent to use. RNA isolation from serum or plasma is tough and we send samples to our genomics core to use a bead robot to get good, reproducible extractions.
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