Hello,
I have problems with RNA extraction from cells. I work with primary neurons and astrocytes cultures, with 1*10^6 cells for each well (more or less, because some cells die).
Actually, I use the Trizol extraction method. With tissue I don't have problems but with cells the 260/280 ratio is horrible, maybe because of Trizol. I don't know. I use the Trizol protocol and I add DNAse in the last step because my probe can react with gDNA.
I use these samples for quantitative PCR, for this reason I need a pure RNA. How can I solve this problem? How can I improve the ratio?
You should use Kit. There are many kits available and the best are Zymogen and MN total RNA extraction kit. You will definitely get good quality RNA with better concentration.
First I would recommend to keep right ratio of cells to Trizol reagent. If you have too many cells and too little Trizol it will affect A260/280 ratio a lot. For I mln cells such as neurons (cells pellet in appr 50ul PBS or directly to culture well after cells wash and residual -appr 50ul of PBS left) I would add minimum 1ml of Trizol. You can use double Trizol method. Isolate RNA by Trizol as usual, then air dried RNA pellet treat with DNAse, then add another 500ul-1 mL of Trizol into DNAse/ RNA mixture (appr. 20-50ul) and isolate RNA again. Also adding glycogen (see instructions in Trizol manual for concentrations) into Trizol itself or into isopropanol helps a lot with higher RNA yields if cell numbers are small. Make sure you never pick up interphase when collect aqueous phase after chloroform addition- better pick up less aqueous phase, then get DNA/proteins/Trizol contamination. Also combination of Trizol/ RNA columns method will help a lot to get pure RNA. After addition of chloroform and collecting aqueous phase you need to add equal volume of 70% (important!, not 100%) ethanol to aqueaus phase and put mixture on RNeasy column, then go through regular column washing steps written in the kits protocol. Also if you have too many dead cells- your A260/280 ratio will be much worse compare to healthy (>90% viability) cell cultures. So you need to wash cells well (multiple times) before pelleting them/ or for neurons I assume you just add Trizol directly to the wells after removing culture medium and washing cells with PBS?. For nonadherent cells I used Ficoll or dead-cell removal magnetic kits to get rid of dead cells- not sure it will work for neurons though. For problematic cells I usually do Trizol isolation first (with isopropanol) , then DNAse I treatment, then add Trizol into RNA/DNAseI mixture again and proceed through RNA columns (as I already described- after equal volume of 70% ethanol added to separated aqueous phase) to get really good quality RNA from problematic tissues. Also keep in mind that coated with Matrigel for example wells for neuron cultures will give a lot of protein contamination, so RNA needs to be purified couple of times to be high quality. Good luck!
You can try RNAeasy kit, you will get A260/280 at value 2.0 or close to 2 for RNA. I have used a lot.
I also prefer column kits - lysis buffer included in the kit plays similar role to Trizol, and you avoid problems with improper purification of proteins, what may happen in "pure" Trizol method.
What I found important:
- use good (proven) kits (which are expensive, unfortunately)
- wash the cells with PBS before lysis and do not overload lysis buffer with the cells, as Natallia Mikhalkevich said
- add a reducing agent (DTT instead of b-mercaptoethanol which is toxic) to the lysis buffer
- do not rely on "RNA-binding affinity of the column", always use DNAse
- let the elution buffer "rest" on the column for 5-10 minuts before last spin.
I work with cell cultures a lot and this works for me.
Natallia Mikhalkevich thanks! I'll try to do all improvements
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