MicroRNAs (miRNAs) are tissue specific, small, non-coding RNAs regulating gene expression, and could represent ideal candidates for early detection of lung cancer. In the featured protocol, we will isolate microRNA from patient blood samples for early lung cancer screening.
1. Plasma Sample Collection
- Collect 10 mL of whole blood sample in Vacutainer tubes with spray-coated K2EDTA and store at room temperature.
NOTE: To minimize the hemolysis, separate plasma within 2 h. Do not store the whole blood at low temperature (i.e., 4 °C) to avoid thermal shock and cell lysis that lead to an unspecific miRNA release.
- Within 1 h, separate the plasma by a first centrifugation step at 1,258 x g and 4 °C for 10 min.
- Transfer the plasma supernatant into a 15 mL tube, carefully avoiding contact with the lymphocytic ring.
- Centrifuge the plasma a second time at 1,258 x g and 4 °C for 10 min.
- Aliquot 1 mL of plasma into 1.5 mL cryovials, avoiding collecting the plasma fraction at the base of the tube.
- Store all the aliquots at −80 °C, except one for the evaluation of the hemolysis. The molecular analysis should be performed within 5 weeks.
2. Plasma Total RNA Extraction
- Prepare a 1-Thioglycerol/Homogenization (OMG) solution with 20 µL of 1-Thioglycerol per mL of Homogenization Solution. Since 1-Thioglycerol is viscous, carefully pipette for accurate measurement. Chill the OMG Solution on ice or at 2-10 °C before using it.
NOTE: The working solution is stable at 2-10 °C for 1 month.
- Suspend the lyophilized DNase I by adding 275 µL of nuclease-free water into the vial and gently mix (do not vortex). As a visual aid, add 5 µL of blue dye to the reconstituted DNase I and dispense the solution into single-use aliquots in nuclease-free tubes. Store reconstituted DNase I at -30 °C to -10 °C.
- Starting from 200 µL of plasma, add 200 µL of the chilled OMG solution.
- Vortex 15-30 s to ensure a complete homogenization. If foaming occurs, let the sample settle on ice.
- Add 200 µL of Lysis Buffer and 25 µL of Proteinase K to the homogenized sample and vortex for 20 s.
- Incubate the samples for 15 min in a thermomixer preheated at 37 °C.
- Load a cartridge for each sample on the deck tray of the instrument and place the plugger in the proper position.
- Transfer the lysate to the appropriate position in the instrument cartridge.
- Add 5 µL of DNase I solution to the proper position in the cartridge.
- Add 60 µL of nuclease-free water to the base of each elution tube.
- Select the "RSC miRNA Tissue" method and begin the automated purification run. The total RNA samples can be stored at −80 °C.
|20xTE Buffer, pH 8.0||Molecular probe||T11493||Dilute the 20X stock solution to have the final concentration of 0,1X in Nuclease-free water|
|Maxwell RSC miRNA Tissue Kit||Promega||AS1460||The kit contains Homogenization Solution, DNase I, Proteinase K, Lysis Buffer and cartridges. For 1-Thioglycerol, inhalation and contact with skin and eyes should be avoided. Use it under a Fume Cabinet.|
|Vacutainer Safety-Lock Blood Collection Set||Becton Dickinson||367286-364815|
|Vortex Mixer||Velp Scientifica||F202A0173|
|ThermoMixer T- shaker||Euroclone||A00564||Bring the Thermo Mixer to 37 °C before starting RNA extraction|
|Maxwell RSC Instrument||Promega||AS4500|
|Cryogenic vials 1.5 ml||Sigma-Aldrich||Z359033|