The miRVANA miRNA Isolation Kit is designed for the extraction of small RNAs, including microRNAs (miRNAs), from various sample types such as tissues, cells, and biofluids.

Dear Giulia Lencioni , it follows the protocol, step by step I did during my doctorate:

kit miRVana PARIS of Life Technologies (Thermo Scientific)

Place in a tube of 100 to 625 μL (6 to 8 volumes per mass of tissue) of the ice cold cell burst buffer;

- Place the tissue in the tube with the beads (0.3 to 0.5g)(this step can be done using a manual homogeneizer tissue also, but without the beads);

- Grind the tissue;

- As soon as grinding place a portion of equal volume of denaturing solution 2x (the high concentration of guanidine thiocyanate in this solution will prevent RNA from being degraded by the ribonuclease) -> the denaturing solution 2x must be at room temperature;

- Vigorously mix the tube;

- Incubate the mixture on ice for 5 minutes;

- Add a volume of acid-phenol: chloroform equal to the volume of the lysed sample + the denaturing solution 2x (make sure to remove the bottom phase, not the aqueous buffer above the mixture);

For example, if the initial lysate volume was 200 μL and was mixed with 200 μL of the 2x denaturing solution, add 400 μL phenol acid: chloroform- Vortex for 30 to 60 second to mix;

- Centrifuge for 5 minutes at> 10,000 rcf at room temperature to separate the mixture in aqueous and organic phase. After centrifugation, the central phase should be compacted, if not, repeat centrifugation;

Step EIII - Carefully remove the aqueous phase (top) and transfer to a new tube. Record the recovered volume;

- Preheat the elution solution or nuclease-free water to 95 degrees celcius to use the filtered RNA at the end of the procedure;

NOTE: The filters can be centrifuged up to 10.000 RCF, centrifuging more than this can damage the filters.

- Add 1/3 of the 100% ethanol volume to the aqueous phase recovered in step EIII and mix vigorously;

- For each sample place one filter in each collector tube;

- Pipette the lysate / ethanol mixture (from the previous step) into the filter;

- Centrifuge for 30 seconds, or until all the mixture passes through the filter;

- Collect the filtrate and place in a new eppendorf. Record the amount collected;

- Add 2/3 of the 100% ethanol at room temperature to the filtrate and mix vigorously;

- Pass the filtrate / ethanol mixture through a second filter, but this time discard the filtrate, reuse the collection tube for the washes;

- Apply 700 μL of the microRNA lavage solution 1 to the filter and centrifuge for 15 seconds. Discard the filtrate from the collection tube, and reinsert the filter into the same tube;

- Apply 500 μL of the 2/3 (this is the name of the solution, not the volume) Wash Solution and extract from the filter as in the previous step;

- Repeat with one second 500 μL of 2/3 Wash Solution;

- After discarding the last wash filtrate, replace the filter in the same tube and centrifuge the set for 1 minute to remove the residual fluid from the filter;

- Transfer the filter to a new manifold. Apply 100 μL of elution / water-free pre-heated nuclease solution to 95 degrees celcius in the center of the filter and close the lid. Centrifuge for 30 seconds to recover RNA;

- Collect the eluate (which contains the RNA) and store at -20 ° C or colder.

I hope that it helps you.

Pedro

Hello Giulia

I am a researcher in miRNA regulation and used miRVana kit to isolate total RNA from monocyte/macrophages.

You should sure to withdraw from the bottom phase in the bottle of Acid-Phenol:Chloroform, because the upper phase consists of an aqueous buffer.

Protocol (Page 11): mirVana™ miRNA Isolation Kit Protocol (PN 1560M Rev C) (thermofisher.com)

Best regards

Hello, Oscar Rafael Escate Chávez, thanks for your answer. I imagine that you do not agitate the solution before using it. Is it correct?