The overall goal of this procedure is to measure the levels of 24 plasma circulating microRNAs in order to determine the risk of developing lung cancer in heavy smokers older than 50 years. These methods can increase cost-effectiveness of a low-dose computed tomography screening programs for early lung cancer detection by reducing the number of false positive nodes and possibly over diagnosis. The main advantage of this technique is that secreting microRNA are easy to measure with standard equipment adopting common molecular laboratories.
We first had the idea for this method when we began to study the role of tumor microenvironment on the development of lung cancer. MicroRNAs reflect the host's response and the dynamic interaction between the tumor and its microenvironment. Visual demonstration of this method is critical to better understand standard operating procedure to follow for this type of molecular analysis.
To start this protocol use Vacutainer tubes to collect 10 milliliters of whole blood sample. Store at room temperature. Do not store whole blood at low temperature, such as at four degree celsius, to avoid thermal shock and cell lysis.
That lead us to a specific microRNA release. Centrifuge the plasma within one hour to separate it. While making sure to avoid contact with the lymphocytic ring.
Transfer the supernatant into a 15 milliliter tube. Then, centrifuge the supernatant at the same conditions. Aliquot one milliliter of this plasma supernatant into 1.5 milliliter cryo vials, being careful to avoid collecting the plasma fraction from the tube bottom.
To start RNA isolation add 200 microliters of pre-chilled OMG solution to 200 microliters of plasma for each sample. To ensure complete homogenization vortex for 15 to 30 seconds. To homogenize the sample, add 200 microliters of lysis buffer and 25 microliters of protein ASK per sample, and vortex for 20 seconds.
Then, incubate the samples at 37 degrees Celsius in a thermo mixer for 15 minutes. Next, select RSC mi-RNA Tissue method and load a cartridge for each sample on a deck tray of an automatic extractor of nucleic acids, properly placing the plunger. Transfer the lysate and add five microliters of DNase one solution to their appropriate positions in the instrument cartridge.
To the base of each elution tube add 60 microliters of nuclease-free water. To begin the automated purification start the run. Store the total RNA samples at 80 degrees Celsius.
To convert the RNA to CDNA using Taq miroRNA reverse transcription kit, use Taq RT primer tool with the mi-RNAs of interest. In a 1.5 milliliter tube placed on ice, prepare RT reaction mix according to the kit instructions. Then add 3 microliters of total RNA per sample, to a final volume of 15 microliters.
Incubate on ice, for five minutes. Finally, load the samples on a thermocycler and run the RT reaction. Then mix 2.5 microliters of each RT reaction product with Taq Master Mix 2X and Custom Taq pool.
Add nuclease-free water to a final volume of 25 microliters. Perform the preamplification reaction in a thermocycler using a specific thermal profile. Then dilute each product by adding 175 microliters of 0.1 XTE PH 8.0.
First, mix 1.13 microliters of the diluted preamplified sample with 2X Taq Universal Master Mix in a 0.5 milliliter tube. And then add nuclease-free water. To measure plasma levels of 24 specific miRNAs on eight samples simultaneously use 384-well microfluidic custom Taq array microRNA card.
Load up to eight reactions with the PCR reaction mix on the card. Centrifuge the custom card at 311 times G, for two minutes and seal it with array card sealer. Finally, place the card in a real time PCR system and run the real time PCR reaction.
To extrapolate and analyze data use appropriate software and obtain raw CT values. Set an automatic baseline to remove the background signals and a fixed threshold of 0.15 for all assays and samples. A preanalytical quality control step has to be performed to identify non analyzable hemolyzed plasma samples in which false results from non specific release of miRNA by blood cells could occur.
A spectrophotometric analysis of plasma samples measuring the absorbance ratio between wavelengths A414 and A375 show the difference between hemolyzed non analyzable samples and non hemolyzed samples. To identify any technical issues, the RT-QPCR performance is evaluated. Four quality microfluidic cards result in RT-QPCR with low performance, and in this case preamplification product should be reloaded to a new microfluidic card.
Good quality microfluidic cards result in good performance, and those are then subjected to two post analytical quality control steps. After performing quality control steps the microRNA signature classifier is applied. The four signatures of miRNA ratios composing the MSC are generated.
Risk of disease, risk of aggressive disease, presence of disease, and presence of aggressive disease. By combining the four signatures, the level of risk is finally defined as low, intermediate, or high. Once mastered, excluding one week of plasma storage at 80 degrees Celsius, this technique can be done in ten hours.
Analyzing eight samples simultaneously. The microRNA signature classifier has been tested so far on more than 10, 000 samples collected from more than 4, 000 volunteers enrolled in the BioMed Lancaster Screening Trial. Potentially these tests could enable greater consistency of the diagnostic algorithm.
They also decreasing screening costs. After watching this video you should have a good understanding, allow to measure circulating miRNA levels in order to use them as biomarker for cancer diagnosis and other severe disease.
