This is the first paper that addresses technical problems in sequencing small RNAs derived from extracellular vesicles from cells. NGS samples require stringent preparation and quality control. Due to the nature of EVs, the QC process of EV samples deviates from the norm.
The advantage of this protocol is the QC steps therein for first-time users. Demonstrating the procedure will be Junyi Su, a research assistant from my laboratory. To begin, plate human mesenchymal stem cells at 2, 000 cells per square centimeter in fresh MSC media in five T175 flasks.
Then grow the cells at 37 degrees Celsius in 5%carbon dioxide environment and replace the media every two to three days, until five flasks of 90%confluent cells are obtained. Next, wash the cells three times with 20 milliliters of PBS per flask. Add 20 milliliters of EV collection media into each flask.
And incubate them at 37 degrees Celsius in 5%carbon dioxide environment for 48 hours. Collect the media and centrifuge it for 10 minutes at 300 g and four degrees Celsius. Then collect the supernatant, and centrifuge the media for 20 minutes at 2, 000 g and four degrees Celsius.
Collect the supernatant again, and centrifuge the media for 30 minutes at 15, 500 g and four degrees Celsius. Collect the supernatant for the last time. Next, transfer the media to ultracentrifuge tubes.
A 60Ti fixed angle rotor is used here, and the pellet will be anchored to the side of the tube. Mark the side of the lid where the pellet is expected, and draw a circle on the side of the tube where the pellet is expected. And pellet the exRNAs for 90 minutes at 100, 000 g and four degrees Celsius.
After the final centrifugation, remove the supernatant. Use small pieces of absorbent paper to dry the inside of the tube without touching the bottom of the tube. And then invert the tube on an absorbent paper.
To resuspend the pellet, add 200 microliters of PBS into each tube, and vortex the tube for 30 seconds. Pipette up and down 20 times. Then assess the extracellular vesicles and other biomolecules with nanoparticle tracking analysis.
And store the pellet at minus-80 degrees Celsius until further experiments. After thawing the samples, use an RNA isolation kit to extract RNA according to the manufacturer's protocol. Elute the RNA from the column provided in the RNA isolation kit in 100 microliters of RNAse-free water.
To concentrate the RNA through ethanol precipitation, add one microliter of glycogen, 10 microliters of two-molar pH-5.5 sodium acetate, and 250 microliters of pre-chilled 99%ethanol into 100 microliters of the purified RNA. Then incubate the samples at minus-20 degrees Celsius overnight to precipitate the RNA. To pellet the RNA, centrifuge for 20 minutes at 16, 000 g and four degrees Celsius.
Next, remove the supernatant, and wash the RNA pellet with one milliliter of 75%ethanol. Centrifuge again for five minutes at 16, 000 g and four degrees Celsius to pellet the RNA. Remove the ethanol, and leave the lid of the RNA tube open for five to 10 minutes to air-dry the RNA pellet.
Resuspend the RNA pellet in seven microliters of RNAse-free water, and use a chip-based capillary electrophoresis machine to detect the RNA quality and concentration. To begin the library construction, pipette five microliters of the RNA into a pre-chilled 0.2-milliliter PCR tube. Next, dilute 3'adapters in RNAse-free water at a one-to-10 volume ratio.
Add 0.5 microliters of the diluted adapter into the RNA tube, and pipette the mixture up and down eight times. Centrifuge briefly to collect all the liquid at the bottom of the tube. Incubate the RNA adapter mixture at 70 degrees Celsius for two minutes in a preheated thermal cycler.
And then place the sample back on the chilled block. Next, add one microliter of the ligation buffer, 0.5 microliters of RNAse inhibitor, and 0.5 microliters of the T4 RNA ligase into the RNA adapter mixture. Pipette up and down eight times, and centrifuge briefly.
Then incubate the tube at 28 degrees Celsius for one hour in the preheated thermal cycler. Next, add 0.5 microliters of the stop solution into the sample tube, with the tube staying in the thermal cycler. Pipette up and down eight times, and continue to incubate at 28 degrees Celsius for 15 minutes.
Dilute 5'adapters in RNAse-free water at a one-to-10 volume ratio. Add 0.5 microliters of the diluted adapter into a separate 0.2-milliliter PCR tube. Heat the 5'adapter at 70 degrees Celsius for two minutes.
And then place the sample on the chilled block. Add 0.5 microliters of 10-nanomol ATP and 0.5 microliters of the T4 RNA ligase to the 5'adapter tube. Pipette up and down eight times.
And centrifuge briefly to collect all the liquids into the bottom. Next, add 1.5 microliters of the 5'adapter mixture into the RNA sample. Pipette eight times very gently, and incubate the sample at 28 degrees Celsius for one hour.
To obtain cDNA, dilute the reverse transcriptase primer in RNAse-free water at a one-to-10 volume ratio. Add 0.5 microliters of the diluted primer into the RNA sample, pipette eight times very gently, and centrifuge briefly. Then incubate the RNA and the primer mixture at 70 degrees Celsius for two minutes in the preheated thermal cycler.
And then place the sample on a chilled block. Next, add one microliter of 5X First Strand Buffer, 0.5 microliters of 12.5-millimolar DNTP mixture, 0.5 microliters of 100-millimolar DTT, 0.5 microliters of RNAse inhibitor, and 0.5 microliters of reverse transcriptase into the sample tube. Pipette eight times very gently.
And centrifuge briefly. Incubate the sample at 50 degrees Celsius for one hour in the preheated thermal cycler. Next, add 4.25 microliters of ultrapure water, 12.5 microliters of the PCR mix, one microliter of the index primer, and one microliter of the universal primer into the cDNA sample.
Pipette up and down eight times, and centrifuge briefly. Finally, place the sample in a thermal cycler, and set up the cycler according to the manuscript. To purify the RNA library, use an automated DNA size fractionator to elute the bands between 140 and 160 base pairs.
Run the extracted library on a column-based PCR purification kit, and elute the library in 10 microliters of RNAse-free water. To check the size of the library, load one microliter of the purified library onto a DNA chip, and follow the manufacturer's protocol. To quantify the library concentration, first dilute one microliter of the purified library in 10-millimolar pH-8.0 Tris-Hcl with 0.05%polysorbate 20 at a one-to-1, 000 volume ratio.
Then run QPCR using the DNA standards of the commercial library quantification kit. Finally, pool equal amounts of the libraries according to the requirements of the sequencing machine, and sequence on a high-throughput sequencing system. The electropherogram of extracellular RNAs included a broad range of RNA.
In contrast, cellular RNA showed very distinct peaks, corresponding to 5StRNA, 5.8SrRNA, 18SrRNA, and 28SrRNA. The resulting quality of both libraries were high and comparable. The length distribution of both libraries showed a single peak at 22 nucleotides, with correlates with microRNAs.
In contrast, the libraries from extracellular RNAs were more heterogeneous, and contained two more peaks which correlate with tRNAs, halves and fragments, or piRNAs. 65%of reads from cellular RNA were mapped to human microRNAs, and each of the other small RNAs accounted for a small portion of the mapped reads. Contrastingly, only 8%of extracellular RNAs matched to human microRNAs.
The most abundant small RNA to which the reads mapped were tRNAs, and the unmatched reads later matched to bacterial sequences. A comparison to the bovine genome suggested that FBS was not a source of contamination. Hence, extracellular RNAs exhibit an atypical profile compared to normal cellular RNA.
Library preparation is a crucial part of this protocol. Mixing must be done very gently to avoid bubbles from forming. This method can also be used to investigate the profile of exRNAs from other kinds of cells, which helps to study the function of exRNAs.
