Dear everyone,
I would like to run an agarose gel electrophoresis to view my RNA integrity. For RNA extraction, I was using the QIAamp RNA extraction, QIAGEN product (Human blood).
Unfortunately, I didn't get any band when I'm using the bleach protocol(denaturing protocol ) and normal agarose protocol (nondenaturing protocol).
Does anyone has any protocols that I can try and hopefully that protocol not using :
1. MOPS
2.miliQ water
3.Fomaladhyde
4.Urea
5.DEPC water
Because I didn't have the following items as I mention above.
Does anyone have an easy and effective recipe for RNA?
Thank you with best regards.
Nur Atiqah
Yes , I did, Sometime I get 1.86, 1.73, 1.76, 1.82, 1.83 and 1.80 for A 260/280 .
It still in good purity. However, I didnt get any band.
Do you have any suggestion?
You can also load 2ug of total RNA mixed with molecular biology grade glycerol into a normal clean electrophoretic device on a 0.8% agarose gel (is better to wash all the items with RNase ZAP and rinse with DEPC water or clean water at least) at the maximum voltage permitted for 10 minutes and check the presence of bands on UV. This is not the best way but is the only solution for you I guess.
I can also say your 260/280 is perfect for DNA but too low for RNA (you should have a value between 1.9 and 2.0), probably you have gDNA contaminations and is better to perform DNAse reaction prior to cDNA syntesis.
Dear Affrida Abu Hassan,
The concentration of RNA in ng/uL and get it around 50 ++ ng/uL.
Dear Paolo Scarpelli, I will try based on your suggestion , And I was perform to eliminate the gDNA contaminations prior to cDNA synthesis for my gene expression in qPCR. Thanks for the suggestion
You're welcome. Don't forget to prepare new clean 1x TAE, possibly from 50X commercial sterile TAE and sterile water. You can also open a new agarose powder pack only for this purpose (this ensure no one used the powder with RNAse-contamined spoons. This can not guarantee RNAse free conditions but help highly to minimize RNAses contaminations.
See the discussion at:
https://www.researchgate.net/post/Can_anyone_help_me_with_gel_electrophoresis_of_total_RNA
Thanks Paolo Scarpelli, by the way do you know the protocol of using gel red staining?
Does it just follow the standard protocol?
Such as load 2ul gel red and 5ul of sample?
When I search about gel red, it state like this:
"
For a 30 mL 1.5% agarose gel:
mix 0.45g agarose + 30 mL 1x TBE
microwave until agarose solution boils and clarifies
let cool to around 50 C
add 3 uL gel red
swirl the mixture
cast the gel (don't forget comb)
This typically works for me. I run the gel in a 1x TBE buffer at around 120 volts."
But, statements as above just explain to add 3ul gel red into agarose gel.
Have you experience performing gel electrophoresis by using gel red? If yes, can you share the protocol?
Dear David f Barker,
Thanks for sharing the link regarding RNA electrophoresis.
From the discussion that have been made by Marwa Khalil Ibrahim, I want to ask something from your answer:
"
Yes, it is not necessary to run a denaturing gel just to check the RNA quality. A freshly prepared 1% agarose gel in TAE or TBE or sodium borate buffer should work fine as long as you are reasonably careful to wash your gel box with distilled water and use distilled water for the buffer. (I would NOT assume that your water needs to be DEPC treated. Good quality distilled water really should be RNAse free.)
The attached photo shows RNAs ranging in quality from excellent (on far right) to degraded (left center) run on a non-denaturing 1% agarose gel. "
Do you have a suggestion how to make sure the quality of distilled water should be RNAse free?
Before this, I just assume the distiled water did not have RNase.
Nur Atiqah Alias:
Yes, I agree with you. I would assume that distilled water does not have RNAse, and use it accordingly. No need to DEPC treat it. I would only investigate the possibility of RNAse contamination if there is a problem encountered. What I have found to be most important is to use freshly prepared gels.
Sorry for late reply. I used gel red in the past instead of ethidium bromide. It works perfectly with both DNA and RNA samples. I'm actually using sybr green with comparable results and the same protocol.
For RNA I prefer a fast run (specially for total RNAs samples) so I use a 0.8% to 1% maximum agarose gel. I boil 1x TAE to dissolve agarose, I let the solution cool a little bit before adding 10 ul in100 ml of gel red or sybr green (I've 10000x stock solutions). So you can just follow the standard protocol
David f Barker:
Yes, I also use freshly prepared gels and make sure the agarose powder has labelled RNase and Dnase Free. Thanks David for sharing your knowledge and idea. I really appreciate.
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