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)  

Thanks Dong or your guidance

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 

Thank you Vu.

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?

Hi Britt

Thank you. I thought about this problem. Recently, I use nuclease-free water for my TAE buffer. The most helpful thing was keeping all the steps on the ice and not room temperature. I also added an incubation in freezer according to other protocol. The extracts are better now. Not perfect but good.