Extracellular MicroRNA Extraction and Validation Protocol

Body Fluid Collection

1. Collect and process the samples in the same manner within the whole study. In case of gender-specific diseases or one-gender cohorts, use the control samples of the same gender. If age-matching is possible, use the samples of patients of the same or similar age.
2. Preferentially use the stabilization tubes or other stabilization chemicals for the sample collection to stop RNase activity in samples. Sample collection into stabilization tubes should be done as rapidly as possible.
3. Collect the samples within the same daytime range, ideally in the morning. Use the fresh void second morning urine. Avoid using the first morning urine.
4. Presurgery/diagnostic samples have to be collected before the surgery (and a treatment application). Consider that all medication applied to patients in the course of sampling may affect the results.
5. Store the collected samples in stabilization tubes either at ambient temperature or between 6 °C and 12 °C (preferentially) for up to 1 month before further processing. Do not freeze.
6. In case of other collection techniques, particularly without stabilization reagents, an immediate sample processing (centrifugations and RNA isolation and storage at -80 °C) should be applied.

Separation of Extracellular Fraction

1. Apply two consecutive centrifugations, at 430 × g (or up to 1000 × g) for 20 min at room temperature and at 2000 × g (or up to 2500 × g) for 10 min at 4 °C. Avoid using stronger centrifugation forces as these can eliminate some proportion of exosomes or other vesicles carrying miRNAs. This two-centrifugation step is necessary to eliminate cells and platelets (blood, ascites), or the potential precipitates and cell/bacterial debris contamination (urine).
2. Transfer the extracellular fractions (supernatants) to PCR-clean tubes, freeze, and store the samples at -25 °C for the short term or at -80 °C for the long term.
3. Inspect hemolysis in the plasma/ascites samples as this is often occurring in cancer samples or in case of suboptimal blood drawing. Hemolytic samples should be avoided (or treated separately) as erythrocyte-derived miRNAs may affect the results. This applies also in case of urine samples should contain red blood cells and hemolysis has occurred.

Isolation of Total RNA

1. Use frozen sample aliquots ideally of an identical volume, e.g., 2 mL and thaw them at ambient temperature. Do not remove potential precipitates in urine samples after thawing as they may contain miRNAs bound with protein. However, do not use the reddish debris at the bottom of tube in plasma samples.
2. Proceed to isolate total RNA.
3. Use the Plasma/Serum Circulating and Exosomal RNA Purification Maxi Kit for RNA isolation from plasma and Urine Total RNA Purification Maxi Kit (Slurry Format) for RNA isolation from urine. Alternatively, use any of the commercially available kits for total RNA isolation ensuring isolation of small RNAs. Store the isolated (and aliquoted) total RNA at -80 °C.

Validation of Individual MiRNAs

1. Perform a reverse transcription of total RNA to cDNA using TaqMan MicroRNA Reverse Transcription Kit and miRNA-specific stem-loop primers. Instead of full-scale reactions, scaled-down reactions up to one-third or one-half of the recommended original input volumes of all reagents and RNA may be used. Additionally, increased amount of RNA may be applied replacing the ddH2O volume, in order to improve resulting Ct values in low-RNA-concentration samples. Use the same input RNA volume for all samples in the experiment.
2. Prepare the reaction mix on ice. In scaled-down reactions (7.5 μL), each reaction should comprise 2.08 μL nucleasefree water (ddH2O), 0.75 μL 10× reverse transcription buffer, 0.095 μL RNase-Inhibitor (20 U/μL), 0.075 μL 100 mM dNTPs with dTTP, and 0.5 μL MultiScribe Reverse Transcriptase (50 U/μL), prepared in large volume as a master mix. For each miRNA-specific master mix, add 1.5 μL of RT primer (5×) and finally add 2.5 μL of specific RNA sample for each reaction. Mix gently by inversion (do not vortex) and centrifuge briefly.
3. In a thermal cycler, perform the reverse transcription reaction in 0.2-mL PCR tubes with following the conditions: 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. Hold at 4 °C. The RT product (cDNA) may be stored at -25 °C at least for 2 weeks prior to the qPCR.
4. Use 96-well PCR plates according to the qPCR cycler, three wells per sample, i.e., one sample in triplicate.
5. Combine the samples of compared different groups (e.g., cancer vs controls) on one plate when possible, ideally for investigated miRNAs and also the endogenous control(s) necessary for normalization.
6. No-template controls (only master mix with specific primer without cDNA) should be included.
7. Perform scaled-down (10 μL) qPCR reactions. Prepare a reaction premix consisting of TaqMan Universal PCR Master Mix (2×) or Xceed qPCR Probe 2× Mix HI-ROX buffer (5 μL), nuclease-free water (3.8 μL), and miRNA-specific small RNA assay (20×) (0.5 μL) (volumes are per one reaction; three reactions should be per sample), including excess volumes (15%) for pipetting losses.
8. Aliquot reaction premix in tubes for triplicate reactions (i.e., 32 μL with a reserve); Add cDNA product (7.5 μL) from the reverse transcription for each sample by gentle pipetting. Centrifuge briefly.
9. Aliquot 10 μL of premix with cDNA in triplicates for each sample into the MicroAmp Optical 96-Well Reaction Plate and seal it. In case of employing Bio-Rad CFX Connect, use PCR plates, 96-well, non-skirted, and Microseal 'B' PCR Plate Sealing Film.
10. Centrifuge the plate (e.g., at 700 × g to 1300 × g) using appropriate centrifuge and rotor (e.g., Eppendorf 5430) for 1.5 to 3 min.
11. Run real-time PCR reactions using a qPCR thermal cycler (e.g., 7900HT Fast Real-Time PCR System or Bio-Rad CFX Connect). Use the following thermal cycler parameters: 50 °C for 2 min and 95 °C for 10 min. For 40 cycles, use the following: 95 °C for 15 sec and 60 °C for 1 min (or 30 sec in fast mode).

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