Step 1: Cell/Tissue Homogenization
![split_workflow01 split_workflow01](https://www.lexogen.com/wp-content/uploads/2015/05/split_workflow01.jpg)
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.
![split_workflow02 split_workflow02](https://www.lexogen.com/wp-content/uploads/2015/05/split_workflow02.jpg)
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
![Workflow_Split-03 Workflow_Split-03](https://www.lexogen.com/wp-content/uploads/2020/05/Workflow_Split-03.png)
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.
![split_workflow04 split_workflow04](https://www.lexogen.com/wp-content/uploads/2015/05/split_workflow04.jpg)
After centrifugation the gel acts as a seal between the phases.
![split_workflow05 split_workflow05](https://www.lexogen.com/wp-content/uploads/2015/05/split_workflow05.jpg)
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
![split_workflow06 split_workflow06](https://www.lexogen.com/wp-content/uploads/2015/05/split_workflow06.jpg)
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).
![split_workflow07 split_workflow07](https://www.lexogen.com/wp-content/uploads/2015/05/split_workflow07.jpg)
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.
![split_workflow08 split_workflow08](https://www.lexogen.com/wp-content/uploads/2015/05/split_workflow08.jpg)
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
![split_workflow09 split_workflow09](https://www.lexogen.com/wp-content/uploads/2015/05/split_workflow09.jpg)
10 – 50 µl of Elution Buffer or Storage Buffer are added to the silica membrane to elute the RNA bound to the silica carrier.
![split_workflow10 split_workflow10](https://www.lexogen.com/wp-content/uploads/2015/05/split_workflow10.jpg)
RNA extraction is finished and the RNA is ready for any downstream application.