RNA was extracted with Trizol using glycogen and high salt precipitation solution (plant cambium cells laser micro dissected with high polysaccharides contamination).
Results when using glycogen showed around 40 ng/µl, but a peak at 270 nm. Without glycogen showed around 3 ng/µl, but still a peak at 270 nm.
Also in both cases there was a low 260/230 ratio 0.4 - 0.7.
The peak at 270 nm represents phenol contamination, so I performed a second chloroform phase separation, with no variation in amount neither decreased the 270 nm peak.
Then for washing the high salt precipitation solution, I performed a sodium acetate precipitation (3 M Na Acetate) following ethanol wash. Samples were lost.
Any suggestions please?
What's your downstream application? We've done RT-PCR with some pretty weird samples, but it still works. Do a downstream application on a dilution of your sample. You might be diluting the effects of contaminants on your enzymes, too.
I would try an ethanol precipitation. Add 0.1x volume of 3 M NaOAc pH 5.2 and 2.5-4x volumes of 200 proof ethanol. Mix and incubate at a temperature between 4C to -80C for 30 min to a few hours and then spin for 15 - 30 min at max speed at 4C. Rinse the pellet with cold 70% ethanol and then air dry.
Glycogen is a carrier molecule that doesn't improve precipitation yields of RNA. However, it will help you identify the RNA pellet in the tube by increasing its size and turning it a white color.
Miss Barbara
Thank you so much for your answer. Yes, the downstream is RT-qPCR, but to check the quality of RNA first I perform RT-PCR, then dilute the cDNA into RNase free water 1:9 and run PCR 2 times.
The dilution that you recomend, usually in how much volume is set?
Thank you
Mr. Matthew
Thank you so much. I performed your washing method in a 10 mg samples (control experiment). Treated with glycogen.
"A": Trizol method without using glycogen
"B" and "C": Trizol method using glycogen
"W": no sample using glycogen
The "sodacet" word attached to A, B and W means the results after the 3M Sodium acetate wash. Then I ran RT-PCR and PCR (with a housekeeping gene), with "A sodacet", "B sodacet" and "W sodacet" samples. I got clear bands in "A Sodacet" and "B sodacet", but "A sodacet" shows less intensity than "B sodacet".
Here I attach the nanodrop graphs. Note that the "W sodacet" sample graph looks so similar to "A" samples. Also the peak is still high at 270nm in "A" samples.
Thank you
Mr. David
Thank you so much for the information. I really think what you wrote is correct. I was reviewing the articles that you sent to me and I think these CETAB or CTAB method is the clue method for many plants. But I was looking for if I can use this method with laser microdissected plant cells (10 000 cells approx.), I still cannot find a CTAB-laser microdissection cells protocol or article.
Thank you so much for your time.
Mr, David
Sorry for the late reply, finally I got the RNA by the trizol method and concentrate by Sodium acetate precipitation. But the quality was very low, despite I was able to run PCR, for Laser microdissection tissues, samples were lost.
I tried a RNAqueous micro kit and got good results. Thank you for your advice and experience. God bless you.
Diego
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