I am extracting RNA from barley leaves and my gel photos are all like these ones. The problem is that other people used the same method for this plant and they look to not have a problem with that. I have tried to be more cautious about cleanness and RNAse issue, but it did not help as well. Do you have any idea what the problem can be?
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Barley leaves (snap-frozen) were crushed in a microcentrifuge tube using a sterilized metal rod. Approximately 100 mg of the crushed tissue was mixed with 1 mL of TRIsure and incubated at room temperature (RT) for 5 minutes. 200 μL of 24:1 chloroform: isoamyl alcohol was added to the above suspension and the tube was shaken vigorously for 15 seconds and then incubated at RT for 3 minutes. The mixture was centrifuged at 4⁰C at 14,000 xg for 15 minutes and the colourless supernatant was transferred to a sterile microcentrifuge tube containing 500 μL of isopropanol. The tube was held at RT for 10 minutes and then centrifuged at 4⁰C at 14,000 xg for 10 minutes. The RNA pellet so formed was washed with 75% ethanol (made with DEPC-treated water) and centrifuged at 4⁰C at 4,000 xg for 5 minutes. The pellet was air-dried and then dissolved in 33 μL of DEPC-treated water (Sambrook and Russell 2001).
Hi Zaynab,
Have you checked the RIN value for these RNA samples using something like an Agilent Bioanalayzer? Or simply assessed using a spectrophotometer to obtain 260/280 and 260/230 ratios? It looks like your RNA could be degrading a little.
When you crush your barley leaves, do you allow them to thaw out whilst they're being crushed? If so, it could be that this is allowing native RN'ases to become active. If this is the case, you could overcome this by crushing the leaves using a pestle and mortar where you place a small quantity of liquid nitrogen in the mortar while you crush.
Something else you may wish to try - we've been having problems obtaining good quality RNA from some samples. We found that increasing the centrifugation time after adding chloroform to TRIzol to 30 minutes allowed better separation of the aqueous phase. Some poor quality RNA is caused by phenol carry-over and this helped to reduce this.
Finally, how certain are you that your DEPC treatments have worked? Do you test for nuclease activity following treatment? I have found that purchasing certified RN'ase-free water has improved my RNA preps.
Thanks
Mark
Hi Zaynab,
Have you checked the RIN value for these RNA samples using something like an Agilent Bioanalayzer? Or simply assessed using a spectrophotometer to obtain 260/280 and 260/230 ratios? It looks like your RNA could be degrading a little.
When you crush your barley leaves, do you allow them to thaw out whilst they're being crushed? If so, it could be that this is allowing native RN'ases to become active. If this is the case, you could overcome this by crushing the leaves using a pestle and mortar where you place a small quantity of liquid nitrogen in the mortar while you crush.
Something else you may wish to try - we've been having problems obtaining good quality RNA from some samples. We found that increasing the centrifugation time after adding chloroform to TRIzol to 30 minutes allowed better separation of the aqueous phase. Some poor quality RNA is caused by phenol carry-over and this helped to reduce this.
Finally, how certain are you that your DEPC treatments have worked? Do you test for nuclease activity following treatment? I have found that purchasing certified RN'ase-free water has improved my RNA preps.
Thanks
Mark
Thank you Mark. 260/280 ration is around 1 measured by nanodrop (I know it is not very precise!). I do not allow thawing when crushing the seeds, I do all in liquid nitrogen and immediately add TRIsure. I have not done any test for nuclease activity of my DEPC-treated water. I followed the protocol which is so common ( 1% DEPC, incubation overnight and autoclaving). Thanks for suggestions.
Hi Zaynab,
A 260/280 ratio of 1 is very low (should ideally be between 1.8 and 2.0). This could be suggestive of either carry-over of organic solvent (phenol, isopropanol or ethanol) at any step in the preparation. I would try centrifuging for longer once you've added chloroform and see if that helps? Hope you get this sorted out!
You have DNA contamination too. You should perform the RNA extraction at 4C and not at RT in order to minimize the contaminants. I know it is written in the protocol to do it at RT, but in my case always worked better at lower temperatures.
Thanks Marta. I can hopefully solve DNA contamination problem with DNAse treatment.
To reduce the contamination of organic solvent ( to increase the 260/280 ratio), I often perform the additional CI (or chloroform) treatment (one more time) and remove (dry) 70% EtOH well.
1. sample + 1 ml trizol
2. vortexing 2 min
3. add 200 ul CI or chloroform
4. vortexing 2 min
5. centri- 13200 rpm 10 min 4'C
6. 500-600 ul supernatant + chloroform (same vol. of sup.)
7. vortexing shortly
8. centri- 13200 rpm 10 min 4'C
9. 400 ul sup. + 400 ul isopropanol + 40-200 ul 3M NaOAc (pH5.2)
10. 70 % EtOH wash (twice)
Hi there,
In my opinion, it is not simple to figure out your specific problem without seeing your performance directly. I suggest, you follow strictly your protocol and perform carefully in each step to figure out. However, it appears to me that, there is one possibility is that the chloroform might cause the problem (if during 10 min incubation after you add it in the color turned to yellow). I also suggest you should use your own materials (Trizol, isopropanol, EtOH, make sure that they are all clean- it is very important) don't use with others. By doing this, you can control all the steps and your performances. For sure, follow your protocol strictly, you will figure it out easily. Good luck
From the Pic I guss you might use too much plant material. I suggest you use less plant materil,
In my opinion from the pic..
1. Check the RNA loading dye
2. % agrose?
3. Check the ph ( Buffer)
4. worked at lower temperature and changing glove after touching skin or ...
i am sure follow your protocol strictly and hope you will figure it out easily....
Hi,
degradation might also occur during the electrophoresis step, do you make the gel and running buffer also RNAse-free?
Given the low 260/280 value the cause is probably contamination as suggested before.. Did the extra chloroform step help?
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