The overall goal of this procedure is to identify and quantitate microRNAs present in exosomes obtained from blood or cell culture media. This is accomplished by first purifying exosomes from blood or cell culture media using differential centrifugation. The second step is to purify total exo omal RNA, using a nirvana micro RNA isolation kit.
After confirming the integrity and concentration of the purified RNA, it is converted to CDNA using reagents in the Megaplex reverse transcriptase reaction kit. Next, a pre amplification step is performed using Megaplex preamp primers and attack man preempt master mix. To amplify the microRNAs, the final step is to run a realtime PCR reaction using 2 384 well microfluidic cards or TAC man low density array cards containing dried TAC MAN primer probes.
Ultimately, this procedure will enable the detection of up to 758 known microRNAs. This technique measures variations in exo omal micro RNA content that are dependent on the source and physiological conditions under which they're secreted. The implications of quantifying these unique signatures extend toward development of biomarkers and novel therapeutic interventions.Strategies.
To begin this procedure, invert an EDTA coated tube containing 10 milliliters of whole blood five times to ensure mixing of the anticoagulant agent and place it upright at room temperature for 10 to 60 minutes, then centrifuge the sample at 2000 times G and four degrees Celsius for 15 minutes to separate the blood. After 15 minutes, remove the tube from the centrifuge and collect the plasma in a new 15 milliliter tube. Place the plasma on ice and dilute it with an equal volume of PBS.
Then gently mix the sample and place it into the centrifuge centrifuge at 2000 times G and four degrees Celsius for 30 minutes. Next, remove the sample from the centrifuge. Transfer the to a new centrifuge tube.
Add one XPBS to 24 milliliters and centrifuge at 12, 000 times G and four degrees Celsius for 45 minutes. Then transfer the supernatant to an ultra centrifuge tube and centrifuge at 110, 000 times G and four degrees Celsius for two hours. After two hours.
Remove the sample from the ultra and remove the supernatant carefully with a pipette. Resuspend the palate in chilled PBS and place the sample back into the ultracentrifuge. Spin at 100, 000 times G and four degrees Celsius for one hour following centrifugation.
Remove the supernatant carefully with a pipette and resuspend the palate in 300 microliters of RNA lysis buffer. Alternatively, the sample can be suspended in PBS or RIPPA buffer. For electron microscopy and western blot analysis respectively, prepare the area for isolating RNA by wearing personal protective equipment, wiping surfaces and pipettes with R a's zap and using sterile filter tips and tubes.
Then determine if pre amplification of RNA is necessary by quantifying the isolated RNA with a spectrometer for samples with greater than 350 nanograms of RNA preamp amplification may not be necessary unless the transcript of interest is in low abundance. Next, prepare CD NA from purified RNA using megaplex pool's. Reverse transcriptase primers.
Start by thawing the primers on ice. Pool A and pool B contain primers for the micro RNAs on microfluidic cards array A and B while they thaw label two RNAs free 1.5 milliliter tubes for the reverse transcription with pool A primers or pool B primers and prepare master mix for each separately from the components as described in the text protocol. Be careful to keep the pool A and pool B primers separate.
Then invert the tubes with the master mix six times and centrifuge briefly pipette 4.5 microliters of the RT reaction Mix into each tube of the micro ramp eight tubes strips. Next, add three microliters of the RNA sample containing between one and 350 nanograms of RNA to each tube. Stir the sample with the pipette tip and cap the tubes.
Spin the samples down briefly and incubate them on ice for five minutes. After the incubation, load the reaction tubes into the PCR machine and run it under the following conditions. When the PCR has completed, store the CDNA at negative 20 degrees Celsius for up to one week or continue to pre amplification for samples with less than 350 nanograms.
Or if the transcript of interest is in low abundance, perform the following preem amplification steps Begin by following the preamp primers for pool A and pool B on ice and invert them to mix. Next, swirl the tack man preempt master mix, and keep it on ice. Also, then prepare the amplification master mix.
Next pipette 2.5 microliters of the CD NA product into micro amp eight tube strips, and then add 22.5 microliters of the preamp amplification reaction master mix. Finally, incubate the tubes on the ice for five minutes. Then load the sample into the PCR machine and run it under the following conditions.
After the preem amplification reaction is finished, briefly centrifuge the tubes and then add 75 microliters of 0.1 x tris, EDTA pH 8.0 to each tube. Next, mix the tubes briefly and spin them down. Continue on to load the tach man micro RNA array or store the diluted product for up to one week at negative 20 degrees Celsius.
To prepare the samples for the tac MAN micro RNA array, combine 450 microliters of the tac man universal PCR master Mix nine microliters of the diluted preamp amplification product and 441 microliters of nuclease free water to an RNAs free 1.5 milliliter tube on ice. Then mix the tube by inverting it six times and centrifuge the sample. Briefly dispense 100 microliters of the PCR reaction mix into each port of the tac man micro RNA array card.
Next, centrifuge the card twice at 1000 times G for one minute, and then seal the card. Next, load the array into the thermocycler. Next, run the samples using the default thermal cycling conditions provided with the primers in the Applied Biosystems manual for the 384 well TAC Man low density array in order to run mRNA analysis in the applied Biosystem 7, 900 HG fast real-time PCR system.
First, change the heat blocks to run a 96 well plate. The transmission electron microscope image shown on the left is of exosomes isolated from mouse blood. Using the methods described in this video.
The image on the right is of exosomes purified from cell culture media and then gold labeled anti CD 81 exosomes typically range from 30 to 100 nanometers in diameter. The relative abundance of normalized microRNAs found in exosomes isolated from rodent blood, human blood, and human aortic endothelial cell culture Media are shown here as delta CT values normalized to U six RNA endogenous control positive delta CT values indicate that the micro RNA is less abundant and negative. Delta CT values indicate that the micro RNA is more abundant.
Not all microRNAs are present in each sample type. For example, mere 1 26 was not detected in rodent blood exosomes and mere 200 C is absent from H-A-O-E-C exosomes. The remaining four microRNAs are present in all three samples in varying amounts.
Although this protocol focuses on characterizing exo omal microRNAs, individual Mr.NA transcripts can also be quantified from exosomes using QPCR when compared with Mr.NA from whole blood. Exo Omal, RNA showed similar expression of both TNF Alpha and vascular endothelial growth Factor A.Combining these techniques has paved the way for researchers in the field of RNA biology to explore the function of circulating RNAs. In mediating gene expression changes and in identifying molecular signatures associated with exosomes in both normal and disease states.
