Hi everyone,
I extract total RNA for my sample (mouse cell culture) then convert to cDNA and PCR, concentration and A260/280 is 300 ug/ml and 1.97, I feel it quite normal, but no bands even for gapdh after PCR. So I checked my mRNA integrity by running it on a 1% agar gel (TBE), lucky for me, I got 2 bands, the upper one is twice size of the lower one, however, on promega and thermofisher's web, they said the 28/18s RNA should be 4.5K/2K, but the bands I have is 2K/1K, which is about the half of normal size. I got quite confused.
Does anyone have any ideas?
Monica
In agreement with Syed Ali,
Formaldehyde denaturing agarose gel for RNA would tell you better the sizes/MW of the bands (but you have to use a lot of RNA for these (up to 5 or 10 ug/lane).. For the PCR phase, be sure to try different concentrations of cDNA - too much cDNA can inhibit the PCR. Try full-strength cDNA, 1:10 cDNA, 1:50 cDNA and 1:100 cDNA and you will see what I mean. Less is often more here. Perhaps you already have a rough visualization of your RNA integrity (which seems to be good) albeit by running the incorrect gel format. So, doing the dilution study of your cDNA would seem to be the next order of business.
In agreement with Syed Ali,
Formaldehyde denaturing agarose gel for RNA would tell you better the sizes/MW of the bands (but you have to use a lot of RNA for these (up to 5 or 10 ug/lane).. For the PCR phase, be sure to try different concentrations of cDNA - too much cDNA can inhibit the PCR. Try full-strength cDNA, 1:10 cDNA, 1:50 cDNA and 1:100 cDNA and you will see what I mean. Less is often more here. Perhaps you already have a rough visualization of your RNA integrity (which seems to be good) albeit by running the incorrect gel format. So, doing the dilution study of your cDNA would seem to be the next order of business.
HI Monica
In agreement with Jack M Gallup, you should first dilute cDNA samples. Another tip is that you should check the size band on DNA using NCBI-PRIMER balsat (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) maybe the biger bands be related to the genome and smaller bands be related to the cDNA.
Regards,
I agree that denaturing agarose gels are better for RNA quality assessment. However, be sure to select a correct marker DNA. Comparison of ssRNA with dsDNA ladder/marker may give you results, as noted by you. I expect your RNA to be fine. Run it along with a ssDNA/ ssRNA marker.
best of luck.
Thanks to everyone! I used the normal agar gel, so....not denatured. OMG, probably my RNA is just fine. the problem could be cDNA.
Hello Monica,
in the attachment you will find the protocol for preparing denaturing agarose gels for RNA analysis. I recommend to wear gloves when you prepare these gels (to prevent RNase contamination and as protection against toxic chemicals).
Regards, Christian
Hello Monica
I think you got already quite bit of good answers and tips. I definitely would agree with using the right ladder, use RNA for RNA samples. Denaturation would help but in my experience a 1,5 gel is good enogh for a fast check. Run the gel at low speed.
To rule out that your cDNA procedure is correct try to run a RT-PCR with random hexamer oligos and check for any product. If you do not get products than I should suggest to control your cDNA procedure.
Ciao
Roberto
Did you use Gelred for staing? In this case, The RNA band may migrate faster than using Ethidium bromide.
I think roughly that we can't give you proper sugesstion if we don't have complete protocol, troubleshooting and pictures.However, I think your RNA is good and you can mesure it by nanodrop or bioanalyzer which is the best measurmen that you can do in my opinion but the previouse disscusion have a good suggestion for you rather than the time of sepration is important.best wishes.
Dear,
Have you checked if the species you are studying can have a different size ratio for some reason?Best,
It's just because your Dna ladder is ds on a regular TBE. If you would run a denaturing gel and denature your dna ladder you would see the right sizes. Upper band is staining twice more than the lower one because it is twice longer thus twice the staining. Your RNA is fine if it does not smear on gel.
I am agreeing with you Monica, I don't think is your RNA, the problem probably is your cDNA. Others has suggested to diluted out your cDNA, which I also highly recommend. For a 1ug RNA template, I usually only use 1/20 diluted cDNA as my PCR template. Gapdh is so abundant in most cell types, there should be sufficient amount for you to amplify a nice band (other housekeeping genes to consider are Actb and Hprt). Of course, do include a positive control to ensure your PCR reaction is working. If you still obtain no band with only your samples, you will need to repeat the cDNA synthesis (maybe also include a positive control here as well).
I perfectly agree with Chuan Khai Tan: the problem could be the housekeeping you choose. Try with other housekeeping genes and with a proper positive control. Also cDNA concentration in PCR could be a problem. So try with progressive diluition.
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