This method can help answer key questions in the translational research field, such as the relationship between circulating biomarkers and the patient prognosis. The main advantage of this technique is the chance to evaluate the expression of a panel of circulating biomarkers with an easy and reliable methodology with low amount of input material. The implications of this technique extend toward therapy of cancer patients because it could identify circulating biomarkers useful in decision-making and treatment monitoring of patients.
Also, this method can provide insight into circulating biomarkers analysis. It can also be applied to other systems, such as tissue biomarker expression analysis for molecular characterization of disease. Visual demonstration of this method is critical, as the extraction steps are difficult to learn.
To begin, transfer 400 microliters of the plasma sample into an RNase-free, two-milliliter tube. Then, add 400 microliters of denaturing solution to the tube. Add four microliters of one-nanomolar spike-in.
After mixing the solution, add 800 microliters of acid phenol-chloroform. Vortex the solution for 60 seconds, and centrifuge at maximum speed for 15 minutes. Next, transfer the lysate contained in the upper aqueous phase to a clean tube, taking care not to disturb the interphase.
Measure the volume of the aqueous phase recovered, and add 1/3 volume of ethanol to the lysate. Then, place a filter cartridge into a fresh collection tube. Transfer up to 700 microliters of the ethanol-lysate mixture to the filter cartridge.
Centrifuge the filter cartridge for 30 seconds at 10, 000 times gravity. Next, measure the total volume of the flow-through. Then, add 2/3 of the volume of ethanol to the filtrate, and mix well.
Place a second filter cartridge into a fresh collection tube. Then, transfer up to 700 microliters of the sample to the cartridge, and centrifuge at 10, 000 times gravity for 30 seconds before discarding the flow-through. Add 700 microliters of microRNA wash solution to the filter cartridge, and centrifuge it with a collection tube at 10, 000 times gravity for 15 seconds.
Discard the flow-through, and put the filter cartridge back into the same tube. After this, move the filter cartridge to a new collection tube. Then, add 100 microliters of preheated elution solution to the cartridge.
Centrifuge the filter cartridge at 10, 000 times gravity for 30 seconds to recover the microRNAs. Prepare the polyadenylation reaction mix cocktail according to the text protocol. Transfer two microliters of the sample to each well of a reaction plate.
Then, add three microliters of the reaction cocktail to each well. Vortex and centrifuge the plate briefly to spin down the contents. Next, place the plate in a thermal cycler, and follow the procedure outlined in the text protocol.
Prepare the ligation reaction mix cocktail in a centrifuge tube. Next, add 10 microliters of the cocktail to each well of the reaction plate. Then, mix the contents of the reaction plate on a shaker at 1, 900 rpm for one minute, followed by a brief centrifuge of the plate.
After this, place the reaction plate in a thermal cycler, and follow the procedure outlined in the text protocol. To perform the reverse transcription reaction, prepare the reaction mix in a centrifuge tube according to the text protocol. Then, add 15 microliters of the mix to each well of the plate containing ligation product.
Mix the contents of the plate on a shaker at 1, 900 rpm for one minute, followed by a brief centrifuge of the plate. Next, place the plate in a thermal cycler, and follow the procedure outlined in the text protocol. Then, prepare the miR-Amp reaction mix, and add 45 microliters of the mix to each well of a new reaction plate.
After this, add five microliters of the RT product to each well of the reaction plate. Mix the plate on a shaker at 1, 900 rpm for one minute, followed by a brief centrifuge of the plate. Then, place the plate in a thermal cycler, and follow the procedure outlined in table one of the text protocol.
First, dilute each cDNA sample in TE buffer. Then, prepare the PCR reaction mix in a centrifuge tube. adding 10%of the volume in excess.
Transfer 19 microliters of the reaction mix to each well of the plate. Seal the plate, and centrifuge it briefly. Finally, perform the thermal protocol on an RT-PCR system, as outlined in table two of the text protocol.
Serial dilutions of spike-in control were performed in order to choose which was the best concentration to add in all plasma samples of this particular case series. Thanks to this protocol, it was possible to evaluate all the biomarkers in every single patient, as well as a single biomarker in the overall case series. In this protocol, a panel of angiogenesis-related circulating miRNAs in relation to progression-free survival, overall survival, and objective response rate was analyzed in patients with colorectal cancer treated with a chemotherapy regimen.
Comparing the expression levels of miRNA between baseline and first clinical evaluation, an increase in hsa-miR-155-5p was observed. This increase was associated with shorter PFS and OS.While attempting this procedure, it's important to remember to perform the steps using phenol-chloroform under a fume hood. Following this procedure, other methods like isolation and analysis of circulating proteins or coding RNAs can be performed in order to answer additional questions, like evaluation of a more exhaustive panel of circulating biomarkers.
After its development, this technique paved the way for researchers in the field of liquid biopsy to explore the expression of circulating biomarkers in cancer patients. Don't forget that working with blood and chemical reagents can be extremely hazardous, and precautions such as gloves and laboratory coat should always be taken while performing this procedure.
