The overall goal of this procedure is to isolate exosomes present in plasma of NSCLC patients that are free of non-exosomal micro RNA sources like free circulating micro RNAse using a double enzymatic treatment prior to exosome isolation. This method can help the analysis of micro RNA lipid biopsies to the exosome compartment which will help to elucidate their use as biomarkers. The main advantage of this procedure is the enzymatic pre treatment which eliminates non exosomal sources of micro RNA that could interfere with the analysis.
To begin thaw a one mL plasma sample on ice. Once thawed invert the tube several times to disaggregate any cryoprecipitates that may have formed. Then centrifuge the plasma at 2, 000 G's for 20 minutes at room temperature in order to remove cells and debris.
Collect the supernatant and centrifuge the plasma again, now at 10, 000 G's for 20 minutes. Again collect the plasma supernatant and now add 10 microliters of RNase. Incubate the plasma at 37 degrees Celsius for 10 minutes in a thermoblock heater.
We added the RNase treatment step to the standard key protocol in order to degradate free micro RNAse. Later the proteinase key treatment step including the key protocol was exploited in order to degradate RNA assays and protect the integrity of exosomal micro RNAse. Next transfer the plasma to a new tube, add half a volume of 1x PBS and vortex it.
Then add 0.05 volumes of proteinase case solution vortex the sample again and incubate it at 37 degrees Celsius for 10 minutes. Now add 0.2 volumes of exosome precipitation buffer and mix the sample by inversion. Then incubate the sample at four degrees Celsius for 30 minutes.
After the cold incubation centrifuge the sample at 10, 000 G's for five minutes at room temperature. Take a 20 to 50 microliter aliquot of the supernatant and store it for control purposes. Then aspirate and discard the remaining supernatant.
Resuspend the exosome pellet in the buffer of choice at a volume depending on the downstream analysis. For RNA analysis add specific lysis buffer for RNA extraction. Before starting clean the working area and associated items with the proper reagents to ensure an RNAse free environment.
Prepare your reverse transcription master mix according to the suppliers'instructions. In this example two samples will be processed. For each sample, nine master mix aliquots of seven microliters are prepared, one for each selected micro RNA.
Store the reaction tubes on ice. Now add five microliters of exosomal RNA solution to each reaction tube with a total RNA concentration of 10 micrograms. Mix the samples with gentle pipetting.
Then add three microliters of the appropriate reverse transcription primer to each reaction tube and mix the samples gently with pipetting. Let them incubate on ice for five minutes before proceeding. Now the reaction tubes can be loaded into the thermo cycler.
Run the reaction cycles according to the suppliers'requirements. In this procedure all the micro RNAse must be tested in triplicate for each patient. This includes running triplicates for the negative controls.
Make sure to include at least one reference micro RNA to properly normalize the data as no consensus exists for stably expressed circulating micro RNAse. Previously obtained candidate micro RNAse can be used as a reference. Begin with preparing the PCR mix.
Make one mix for every micro RNA that is going to be assessed. Store the reagents and primers on ice protected from light until they are used. For each PCR well prepare to load 10 microliters of PCR master mix.
With 7.67 mL of nuclease free water, and one microliter of PCR primer, escalate these volumes to the number of wells needed to fill on the plate with a little extra. Now load 18.67 microliters of the prepared mixes to the corresponding plate wells. Then add 1.33 microliters of the appropriate reverse transcription reaction product to the appropriate plate well, filling each well to 20 microliters.
For the negative control wells add 1.33 microliters of water. Now close the plate and centrifuge it in a plate spinner for a few seconds at 300 G's. Using the real time PCR software in the template assign the reporters, assign the target genes, define the reaction volumes, and set the experiment wells.
Now load the plate in a thermocycler and define the reaction parameters according to the suppliers'specifications. Once the run is completed export the data including the CT values to a spreadsheet for analysis. Twelve plasma samples from NSCLC patients and six healthy controls were processed as described.
Exosome markers were analyzed by Western Blot. The markers linked to exosomes, ALIX and TSG101 were present in the samples. To biophysically characterize the samples TEM analysis was performed.
Round vesicles were found ranging from 40 to 100 nanometers. Eight micro RNAs, described to be deregulated in NSCLC by various models were analyzed using RTPCR. The internal control mir 1228 was validated as a stable endogenous control in a different sample set.
It was found that the analyzed micro RNAs are deregulated in the clinical samples from the lung cancer patients compared to controls. This protocol allows to analyze exosomal related micro RNAse from plasma samples, avoiding other compartments that might interfere with your analysis. We recommend to you to use the stored supernatant aliquot as a control in order to monitor the efficiency of the enzymatic treatment.
Liquid biopsies are very important in cancer, specifically in lung cancer because several of the components like CTC or free DNA are used not only today but also for follow up with patients. Exosomes are the new component of these liquid biopsies with important pleotropic activities. One of them for example is direct resistance.
I invite to see this video in order to select and characterize your exosomes in your own lab. Thank you for watching.
