Hi everyone,
I’m using RNAeasy plant mini kit of Qiagen in Arabidopsis. My concentration is good and also 260/289 value, but I’m having troubles with 260/230 in some of my samples I have 0,78 and 1,58. What may I do to improve my 260/230? these samples are to send to RNA-Seq.
If you are using water, consider using nuclease-free water, as it may help improve the 260/230 ratio.
It's important to note that an imperfect 260/230 ratio may not always adversely affect downstream applications like RNA-Seq. If the RNA concentration is good and the RNA integrity is preserved, you can still proceed with RNA-Seq analysis. However, if possible, it is advisable to improve the 260/230 ratio to minimize potential interference from contaminants during library preparation and sequencing.
A simple sodium acetate precipitation of the RNA will clean up your A260/230 ratios. Here is the method I use:
- Add ultra pure water make up RNA samples to 100ul in total
- Add 300ul 100% EtOH & 10ul 3M Sodium Acetate per sample
- Vortex + incubate overnight @ -20oC
- Centrifuge full speed @ 4oC for 30mins
- Pipette off supernatant.
- Add 500ul 70% EtOH + mix
- Centrifuge full speed @ 4oC for 15-20mins
- Pipette off supernatant & air dry for at least 15 mins [check that no EtOH remaining]
- Resuspend in 30ul or 12ul (depending on RNA concentration)
- Enjoy good A260/230 values!
Give it a go, it works really well. Just remember the RNA pellets will be difficult to see (pellet paint can help).
- Badges
- Science method