Step 1: Cell/Tissue Homogenization

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The sample is homogenized in an isolation buffer that is highly chaotropic to facilitate effortless and complete solubilization. The SPLIT for Blood protocol adds a red blood cell lysis step prior to sample homogenization.

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The sample is transferred to a phase lock gel column which contains a special gel matrix that acts as a barrier between the organic and aqueous phase based on the density differences.

Step 2: Phenol Extraction

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Acidic phenol and acidic buffer are added to create a monophasic solution, which is essential for the efficient separation of genomic DNA into the organic phase. Chloroform is added and phases are cleanly separated.

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After centrifugation the gel acts as a seal between the phases.

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The aqueous phase containing the RNA can be easily decanted and no carry-over of the organic phase will take place.

 

Step 3: Purification of RNA Fractions

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Depending on the amount of isopropanol added either the total RNA (1.75x isopropanol) can be extracted or the RNA can be split in a large (0.33x isopropanol) and a small RNA fraction (1x isopropanol to the flow-through of the large RNA fraction).

 

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The RNA is precipitated onto a silica column. For the total RNA the entire RNA will precipitate onto the silica carrier, while for the large fraction RNA with a lower limit of about 150 nt will bind whereas the small RNA will be in the flow-through.

 

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The flow-through of the large RNA fraction contains the small RNAs which didn’t bind to the silica column. After addition of 1x isopropanol it can be precipitated and purified onto a new silica column.

 

Step 4: Elution of RNA

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10 – 50 µl of Elution Buffer or Storage Buffer are added to the silica membrane to elute the RNA bound to the silica carrier.

 

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RNA extraction is finished and the RNA is ready for any downstream application.