FAQs

Yes. miRNA can be extracted from all solid samples using PhaseAll™ and following the standard RNA isolation protocol described in the product manual. You can modify this protocol for extraction of free miRNAs from blood serum as outlined below:

1. Sample Preparation:
   Add 50–250 µl of serum to 750 µl PhaseAll™. Gently invert the tube 5–8 times and incubate at room temperature for 15 minutes.

2. Phase Separation:
   Add 200 µl chloroform, invert for 15 seconds, and incubate again for 5 minutes at room temperature.
   Centrifuge at 12,000 × g for 15 minutes at 4 °C.

3. RNA Precipitation:
   Carefully transfer the upper aqueous phase to a new tube. Add 500 µl isopropanol, incubate for 10 minutes at room temperature, and centrifuge again at 12,000 × g for 10 minutes at 4 °C.

4. RNA Washing and Resuspension:
   Wash the pellet with 75% ethanol, air-dry at room temperature for 30 minutes, and resuspend in 20 µl RNase-free water.

5. RNA Quantification:
   Measure RNA concentration and purity using a NanoDrop™ UV-Vis Spectrophotometer at A260/280 nm and A260/230 nm ratios.

FFPE samples should be prepared as follows:

Deparaffinisation:

Remove the paraffin wax from the FFPE tissue section using a solvent like xylene or a specialized buffer.

Xylene is the most widely used solvent in the deparaffinisation of tissue sections. It dissolves the paraffin wax, enabling it to be removed from the tissue. For effective deparaffinization, the slides are typically immersed in xylene or a xylene substitution for 2–3 cycles, with each immersion lasting between 5 and 10 minutes. Xylene is highly effective but also toxic, so appropriate safety measures – such as working in a well-ventilated space or using xylene substitutes – should be followed.

The first immersion into the xylene bath begins dissolving the paraffin. After 5-10 minutes, the slides are transferred to the next bath. A second xylene bath ensures any remaining paraffin is fully dissolved, further improving the quality of deparaffinisation. Researchers may choose to perform a third xylene bath for thicker or older sections.

Rehydration:

After the paraffin is removed, the tissue needs to be rehydrated to restore its original state. This is done by gradually decreasing the concentration of ethanol, which is critical to prevent tissue damage. This step also helps eliminate any residual xylene that may still be present in the sample.

The rehydration process typically follows these 4 steps:

1. 100% Ethanol: The slides are immersed in absolute ethanol for 5 minutes to remove any remaining xylene.
2. 95% Ethanol: The slides are transferred to a solution of 95% ethanol for another 5 minutes. This step starts the gradual rehydration process.
3. 70% Ethanol: The next step involves placing the slides in 70% ethanol for 5 minutes, which further hydrates the tissue.
4. 50% Ethanol: The slides are then moved to 50% ethanol for 5 minutes to ensure full hydration.

By the end of this step, the tissue has been completely rehydrated and is ready for further analysis.

Extraction:

Follow our RNA, DNA or protein extraction protocol in the specific product manual.