I extracted human embryoid body RNA for PCR analysis using Trizol reagent. RNA conc. is between 50-200 ng/ul, and 260/280 ratio is about 1.7-2.1,so these are really good, but 260/230 ratio is extremely low ~0.3-0.7.
The first time I used GeneJET RNA Purification Kit (ThermoSci), both RNA conc. and 260/230 ratio are lower than when I switch to use Trizol reagent. Can I use these RNA for PCR analysis?
Absorbance at 230 is usually guanidium (270 is phenol). As a chaotropic salt it's obviously not a good thing to have hanging around, and yes, it will definitely interfere with downstream cDNA synthesis.
Good, clean, RNA should have a 260/230 ratio greater than 2, but I've generally found that anything about about 1.7-1.8 is acceptable.
Some guanidium often co-precipitates with RNA (especially if you use isopropanol at low temperatures) but it can generally be removed by simply reprecipitating the RNA. I tend to use the following:
Make RNA up to 500ul with H2O (larger vols make everything easier)
Add 50ul of 3M NaAc, pH 5.5 (+ 10ug of glycogen if you want to maximise yield)
Add 500ul of room temperature isopropanol (room temp is important: cold isopropanol will just co-precipitate your guanidium again), mix well, leave at room temperature for 20mins.
Pellet RNA by spinning for 10 mins at 12-13k rpm in a benchtop (chilled to 4 degrees, ideally), and wash 2x with ice cold 70% ethanol (just gently pipette the ethanol down the side of the tube so you don't dislodge the pellet, use maybe 300-500ul, then spin, remove, repeat).
Air dry 15mins, resuspend in whatever volume you think is appropriate (tailor based on expected yield).
I use the above protocol on any samples with 260/230 ratios lower than 1.7, and while you probably lose about 30% of your RNA in the process, the remaining RNA is invariably of much better quality (260/230 > 2.0, suitable for cDNA synthesis).
Your samples are most probably been contaminated by phenol. Phenol can indeed interfere with your qPCR results. For a pure RNA sample, the A230:260:280 should be around 1:2:1 (form wikipedia). In my opinion you should avoid using these samples. Do the procedure again and during the phenol/chloroform washings carefully avoid phenol contamination of your samples (is better to lose some of your rna than getting more with less purity).
Absorbance at 230 is usually guanidium (270 is phenol). As a chaotropic salt it's obviously not a good thing to have hanging around, and yes, it will definitely interfere with downstream cDNA synthesis.
Good, clean, RNA should have a 260/230 ratio greater than 2, but I've generally found that anything about about 1.7-1.8 is acceptable.
Some guanidium often co-precipitates with RNA (especially if you use isopropanol at low temperatures) but it can generally be removed by simply reprecipitating the RNA. I tend to use the following:
Make RNA up to 500ul with H2O (larger vols make everything easier)
Add 50ul of 3M NaAc, pH 5.5 (+ 10ug of glycogen if you want to maximise yield)
Add 500ul of room temperature isopropanol (room temp is important: cold isopropanol will just co-precipitate your guanidium again), mix well, leave at room temperature for 20mins.
Pellet RNA by spinning for 10 mins at 12-13k rpm in a benchtop (chilled to 4 degrees, ideally), and wash 2x with ice cold 70% ethanol (just gently pipette the ethanol down the side of the tube so you don't dislodge the pellet, use maybe 300-500ul, then spin, remove, repeat).
Air dry 15mins, resuspend in whatever volume you think is appropriate (tailor based on expected yield).
I use the above protocol on any samples with 260/230 ratios lower than 1.7, and while you probably lose about 30% of your RNA in the process, the remaining RNA is invariably of much better quality (260/230 > 2.0, suitable for cDNA synthesis).
@John Hildyard,
Your suggestion worked perfectly! Thank you very much. I think you meant NaOAc though (Sodium Acetate) because that's what I used and it worked. I used pH 5.2, and it still seems to have worked.
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