ADS Biotech mRNA Purification Solutions & Organoid Media FAQ
What is the capacity of the columns?
| Column | Capacity |
|---|---|
| DNA-99-3510 | 2 µg |
| RPC-99-3810 | 50 µg |
| RPC-99-2110 | 600 µg |
| RPC-99-3015 | 2 mg |
Is there a maximum sequence length that can be successfully separated using the columns?
No, there is no maximum as the mechanism of separation remains the same for small and large nucleic acids. However, the larger the sequence is, the longer the retention time will be, and the longer runs will take.
Can I get single base-pair resolution in my separations?
How do I prepare my column for storage?
How long do I need to equilibrate the column when changing temperatures?
The column should be equilibrated with 1:1 Buffer A:Buffer B for a minimum of 30 minutes before preparing for sample injection.
What types of nucleic acids can be separated using ADS Biotec columns?
Any type of nucleic acids can be separated using our columns. Due to the mechanism of separation, the length of the species is not a factor. PS-DVB columns have been used to purify mRNA, cRNA, siRNA, asRNA, miRNA, etc.
Can ADS Biotec columns be used to purify 5 kb long sequences?
Yes, our columns are able to purify RNAs that are 5 kb in length.
Can I load IVT product directly to the columns?
Yes, RNA from IVT reactions can be purified on our columns without preliminary purification.
What is the RNA ladder used to generate chromatograms?
We used the RiboRuler High Range Ladder from Invitrogen.
What are the recommended flow rates and pressures for the columns?
| Column | Flow Rate (mL/min) | Pressure Range (psi) |
|---|---|---|
| DNA-99-3510 | 0.7 – 1.1 | 900 – 1600 |
| RPC-99-3810 | 0.75 – 1.5 | 800 – 1400 |
| RPC-99-2110 | 4 – 8 | 800 – 1400 |
| RPC-99-3015 | 4 – 10 | 800 – 1600 |
Organoids Frequently Asked Questions
What are organoids?
Organoids are miniaturized and simplified models of tissues, typically generated from stem cells, or primary tissue samples. Stem cells are fed specific factors to induce differentiation into the cell type(s) of interest. Organoids are used to more accurately model tissue microenvironments that cannot be recapitulated in 2D cell culture alone.
Are there differences between organoids and spheroids?
Organoids typically consist of multiple cell types to replicate the variety of cell types within tissues and the microenvironment between those cells. Spheroids are typically a cluster of a single cell type that is generated by aggregating the cells on special culture plates/conditions. Spheroids are simpler in nature and do not mimic the extracellular matrix in tissues.
How are organoids established?
Using stem cells, the cells are suspended in a basement membrane extract (BME) and exposed to differentiation factors to replicate the various cell types within the tissue of interest. When starting from primary tissue, the tissue is both mechanically and enzymatically digested to produce small cell clusters. The clusters are suspended in BME and cultured to allow the propagation of the organoids.
How are organoids cultured?
Typically, organoids are cultured within BME domes or layers in multi-well plates surrounded or layered with culture media. The three-dimensional culture in BME more closely mimics the three-dimensional microenvironment found within tissues. BME is porous enough that culture media, growth factors, and signaling factors can diffuse to the cell clusters, and waste can diffuse away from the cell clusters. Tissue specific medias are used so that factors normally found in the tissue microenvironment are present in the culture media to promote growth.
What are BME domes?
BME refers to basement membrane extract, which is a gelatinous mixture of compounds and factors typically found within tissue extracellular matrices and are important signaling factors in cell proliferation and maintaining health. The domes allow for three-dimensional growth of organoids.
What media or supplements are required for organoid culture?
There are some components of media that are universally used; however, the supplements usually vary depending on the tissue type and model organism. Optimization over the course of years contributes to updated composition of media and represents an ongoing effort to continually make the products better.
How long can organoids be expanded in culture?
This is dependent on many factors, including tissue type, culture conditions, initial tissue source health, etc. Organoids can generally be cultured for weeks to months.
Can organoids be frozen for long term storage?
Yes, similar to cell lines, organoids can be frozen and later thawed for later usage. Like cell lines, a cryoprotectant must be used to ensure that the organoids are protected from the formation of ice crystals. Cryopreservation allows researchers to store them for an extended period and revive them when needed, reducing the need for continuous culture and passage.