Hi Marconi

Not sure what your further application is but to me only HBL26 seems slightly Ok. See the attached photo of my gel, I used to identify RNA quality. Its not the perfect gel but the 2 rRNA bands (top: bottom) should give you the ratio of 2, which is perfect. However ratio less than but close to 2 is also acceptable.

What I can see in your gel is mostly smear which is most probably degraded RNA.

I never used DEPC but used RNase free water for making all the solution and gel. I rinsed the glassware with milliQ water and then dry autoclaved.

Also check 260/280 ratio and concentration on nanodrop. I loaded 200ng in each well. I also used similar loading buffer made in lab and heated samples at 70 degree C for 10 min then on ice for 3 min.

I think you need to revisit you RNA extraction procedure. You ladder seems alright so as such gel electrophoresis part looks OK.

All the best.

You need to re-extract and purification .....

Because this electrophoresis is incorrect

regards

Hi Hassan Nima,

Why is it incorrect? What happened?

Regards

Dear

Where is the problem?

Dear Al-Ouqaili,

I would like to know if the RNA is intact and with good quality. I know that it must appear two bands... the length of them, in the figure, are correct?

regards

if you want to know the purity of your sample, you have to use nanodrop-spectrophotometer.

Following

Hope you good luck.

I suppose you may have used denaturating conditions, not specified. From left to right, the first lane seems too loaded or alternatively the migration is deformed may be because of some impurities in the extract which have retained the coloration? ; second lane is correct, the large rRNA being a few underrepresented compared to the small rRNA, the 4-5S band is visible ; third lane, not enough and may be degraded RNA (smear displaced toward small length RNAs) ; fourth lane, presumably degraded, the large rRNA being very underrepresented. This is an encouraging result...

Thank you for your answer and help Dr Levesque!

You are right, the gel was made in denaturating condition (i just used DEPC water to make the gel and treated the plastics and glasswares, but nothing else). However the sample was treated in non-denaturing conditions: it was overheated in 65°C for 15 min and later chilled on ice; after that 2,0 uL of the RNA loading buffer (prepared by me: glycerol 50% + EDTA 1,0mM) was added to the sample which following was put to run.

You meant the "HBL20" sample was correct? About the "HBL26" sample, is it ok? The small 4SRNA band is the first one (from bottom to top), is that right? I haven't quantified the samples, the ones which did not appeared might be an insufficient concentration? The RNA from HBL27 lane seems to be degraded?

Hi Marconi

Not sure what your further application is but to me only HBL26 seems slightly Ok. See the attached photo of my gel, I used to identify RNA quality. Its not the perfect gel but the 2 rRNA bands (top: bottom) should give you the ratio of 2, which is perfect. However ratio less than but close to 2 is also acceptable.

What I can see in your gel is mostly smear which is most probably degraded RNA.

I never used DEPC but used RNase free water for making all the solution and gel. I rinsed the glassware with milliQ water and then dry autoclaved.

Also check 260/280 ratio and concentration on nanodrop. I loaded 200ng in each well. I also used similar loading buffer made in lab and heated samples at 70 degree C for 10 min then on ice for 3 min.

I think you need to revisit you RNA extraction procedure. You ladder seems alright so as such gel electrophoresis part looks OK.

All the best.

Hi Robin,

Thank you very much for your explanation and suggestions.

The reason i used DEPC is to turn the distilled water in RNAase free water; I used this water to make the gel and solution as well as to treat the plastics and glassware - I used H2O2 3% firstly and following I used DEPC water to remove it. So you don't think that the problem is how I did the RNA electrophoresis, do you? That is my doubt, if the RNA was really degraded or the RNA electrophoresis procedure degraded the RNA. But if the reason was the later option, all the samples would be degraded, wouldn't they? And, as you said, the HBL26 seems to be slightly ok, despite the fact the 28S/18S is not 2 (we can notice that visually), unlike your gel that we can easily see that the top band shows a higher intensity than the bottom band.

Regarging the RNA purification, I used miRneasy kit, from Qiagen, I don't know what went wrong. What care do you take when extracting your RNA?

All the best

Robin, how do you treat your electrophoresis tank and the accessories to run RNA? I saw this protocol in a qiagen manual, but I don't know if this is the best manner to treat... And how do you treat your plastics?

All the best.

Hi Marconi

Did you autoclaved DEPC treated water or glassware as heating destroys DEPC and then it doesn't interferes?

Regarding my plasticware (gel tanks and combs), I would simply wash it with lab detergent, soak it in ethanol (100%) and then dry it on lab bench just before casting the gel. Never had any trouble. One of my casting tray became bit translucent after ethanol soaking, probably the plastic was not suitable for ethanol but gel and RNA run was fine.

Pipette Tips- I always used RNase free barrier tips for all RNA work. I wiped the table (Whole area) and pipettes, tip boxes and everything I would touch during RNA handling with RNase erase.

Glassware- cylinders for measuring buffer, flasks for preparing gel etc were all autoclaved and then left it incubator to dry properly before use. You can also bake the glassware before use to be on the safe side.

Do not touch anything related to RNA with bare hands. Also wipe your gloves with RNase erase before touching anything.

I also used miRNeasy kit from Qiagen, works perfect. Although some of my RNA quality did not came out good so I had to repeat the samples.

Another factor behind RNA degradation could be sample handling before extraction. Tissue should never be allowed to thaw before it goes into TRI reagent. I would kept it on dry ice or in liquid nitrogen till I am ready to add tri-reagent.

I have also seen someone spoiling RNA as they were using huge amounts of tissue. Make sure you use the appropriate/recommended amount of tissue. Tissue overloading can muck up the RNA as well. Not sure how but it does. I used 100mg for adipose tissue and 10mg for muscle.

Hope it helps. All the best.

Cheers.

Dear Marconi,

using a agarose gel to assess the quality of your RNA is a nice and easy way, however, I would recommend to use capillary gel electrophoresis such as the Bioanalyzer or the Fragment Analyzer. These devices give you an exact measure of your RNA quality and you could rule out if your electrophoresis preparation caused the degradation of your RNA (I doubt that, btw). I never treated my RNA or any of the used equipment before I run my gel electrophoresis and I always got nice distinct bands if my extraction worked. I would also recommend to do a nanodrop measurement to see if you have any contamination in your samples. My guess is that during the extraction the RNA got degraded.

Good luck!

Hi

Yes I agree with the answers that say, HBL26 is a good result which include the intensity on gel of 2 bands 28S rRNA : 18S rRNA is 2:1, the purity (260/280) is 2.

But in HBL20 theres degradation for sure.

I dont think that the degradation i related to agarose preparation or overheating of the gel during the run ,otherwise all samples should be degradate, i think degradation is during extraction or while loading your sample... i hope you get good results next time.

Artur Burzynski might be onto something; what is your ladder? Is the gel formaldehye or glyoxal based denaturant?

Hi Dr Burzynski and Dr Shelton, I just wrote the opposite, the gel i made is non-denaturing and the sample was treated in denaturing condition.

Hi Dr Rana al obaidi, thank you. I thought exactly that, something occurred with the HBL20 sample which caused its degradation.

Dear Dr Robin Wilson and Dr Martin Rippin, thank you for your answers and suggestions.

I actually used the DEPC water in a wrong way, after autoclaving.

All the best.