Encyclopedia of Experiments: Cancer Research
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Transcript
- MicroRNAs, or miRNAs, are small non-coding RNAs involved in gene regulation. Their expressions are altered during cancer, and hence are potential cancer biomarkers. To begin miRNA isolation, take freshly collected whole blood sample and centrifuge at 4 degrees Celsius to separate plasma from blood cells. Transfer the supernatant to a fresh tube and centrifuge again to obtain a clearer plasma supernatant. Extract the supernatant, treat with chilled homogenisation solution, followed by lysis buffer to homogenize the sample. Add proteinase K enzyme solution to degrade proteins, particularly the nucleases that would otherwise digest RNA during downstream steps.
- Next, load the lysate into the designated well of a preassembled instrument specific nucleic acid extractor cartridge. Add DNase 1 solution to the appropriate well to eliminate genomic DNA from the sample. Add nuclease-free water to the elution tube assembled on the cartridge for conditioning before miRNA isolation. Load the cartridge assembly into the nucleic acid extractor. Select and run the appropriate miRNA purification software. When complete, remove the elution tube containing miRNA and store at minus 80 degrees Celsius.
- In the following protocol, we show miRNA extraction from patient blood samples for lung cancer screening.
- 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 4 degrees Celsius, to avoid thermal shock and cell lysis that lead to a non-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 1 milliliter of this plasma supernatant into 1.5 millimeter cryovials, 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 homogenisation, vortex for 15 to 30 seconds. To homogenize the sample, add 200 microliters of lysis buffer and 25 microliters of proteinase K per sample and vortex for 20 seconds.
- Then incubate the samples at 37 degrees Celsius in a thermomixer for 15 minutes. Next, select RSC miRNA 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 5 microliters of DNase 1 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 minus 80 degrees Celsius.
