Hello All,
I'm doing RNA extraction from cells. I tried both ways by using TRIZOL (with chloroform, and isopropanol) and RNeasy mini kit.
When I use TRIZOL, the RNA concentration is high, however the quality of RNA is not good. The ratio of 260/280 is less than 1.8.
When I use RNeasy mini kit, the RNA concentration is low, however the quality of RNA is good. The ratio of 260/280 is more than 1.8.
Can anyone advise me how to get high RNA concentration with good quality together.
Thank you
You can concentrate RNA using ammonium acetate, isopropanol, and ethanol precipitation. However, if your yields from the kit are low, you may want to troubleshoot there instead; make sure all of your buffers are prepared correctly and that you're not overloading your columns.
Hello Elham Amini. E.A
You may focus on the following steps that could help improve the yield as well as the quality of RNA.
1. The sample volume should not exceed 10% of the TRIzol volume. When you exceed the amount of sample, it will result in poor quality phase separation. If you use more amount of sample than the recommended ratio, then the contaminants like proteins and lipids will enter your aqueous phase and contaminate the RNA because not enough phenol and chloroform is available to solubilize these biomolecules.
2. Add 0.75 mL of TRIzol reagent to 5– 10 × 10^6 cells.
3. Do not wash cells before addition of TRIzol reagent to avoid mRNA degradation.
4. If the starting sample is small (
Quality is usually based on careful handling of the starting materials (e.g. keep flash-frozen or stored in an appropriate solution).
Quantity is usually based on the amount & type of starting material and how well it is disrupted. The best is grinding in liquid nitrogen. For tough materials (insects, plant tissues, certain strains of bacteria), you will also want to use a "shredder column".
Read the kit protocols in detail - there are LOTS of variations depending on the type of starting materials. Choosing the right amount of starting material & adding in any suggested "optional" steps will help a lot.
Good luck!
Dear John Hardy Lockhart. Thank you for your reply.
Actually, when I'm extracting RNA with TRIZOL, I'm also using sodium acetate, glycogen, and isopropanol. Through this way, the RNA concentration is good, however, the the quality and ratio of 260/280 is low. My problem is, how to increase the quality as well.
Dear Malcolm Nobre, thanks for your reply.
Actually, I'm using 1 ml of TRIZOL for 5– 10 × 10^6 cells, and also using sodium acetate, glycogen, and isopropanol. and wash my pellet with 75% Ethanol as well. The RNA concentration is good, however, the the quality and ratio of 260/280 is low 1.8.
I used spray RNaseZap onto the surface and changed my gloves. Used the cooling centrifuge and ice as well.
Sorry besides these, I have some question.
-What is the reason for incubate in a water bath or heat block set at 55–60°C for 10–15 minutes?
-I I want to use the combination of TRIzol extraction and washing step and using column from available kits, in which step I can transfer my RNA solution to the column and start the washing step by the kit's reagnets?
Thank you so much
Dear Katie A S Burnette, thanks for your reply.
Actually, I extracted RNA from cell cultures, and follow the protocol from the kit related to RNA extraction from cells. My problem with using kit is, the concentration decrease when I use the column of kit. But the quality is good. I don't know how to increase the concentration as well.
Thank you
Few suggestions from my side
*Use a high-quality RNA extraction kit
*Store RNA properly
*Avoid RNase contamination