5 replies to this topic

[ram]

This is 1% agarose picture of plant RNA. How is this RNA appearing?...is there any sort of degradation? I have read somewhere that 28s rRNA band should be brighter than 18s rRNA (which is not the case with this pic (?)), otherwise it indicates degradation. Is that true?



Below is the RNA isolated from different stage of tissue...where 28s > 18s



Thanks
Ram

Edited by ram, 01 May 2010 - 07:17 AM.

[pDNA]

the RNA appears somehow degraded ...indicated by that smear.

Its hard to tell how much further processing is affected by taking a look at an agarose gel (if you use different exposure the appearance might differ).

We always use the Agilent Bioanalyzer to check the integrity of the RNA ...it gives you a RIN number that measures the quality of the RNA ...the higher the better ...if it is below 7 we do not use the samples for reverse transcription.

How are you going to further process your RNA? ...if am i honest ...my personal feeling towards all this RNA quality stuff is that the efforts made are somehow exaggerated. It always depends on the steps downstream the RNA isolation to what extend the RNA quality is a critical factor.

Regards,
p

[ram]

Thanks for the comments p
I am going to use this RNA for reverse transcription followed by real-time PCR

[pDNA]

you can go through that paper! maybe it helps you a bit!

I think the most important thing is to take care in the analysis of your results (normalization!!).

Good luck!

Regards,
p

Attached Files

•  fleige_pfaffl_rin_2006.pdf   226.39K   118 downloads

[lihkin]

The RNA quality looks somewhat Ok. I also think that your agarose gel has some problem with solidifying/buffer since it appears a little uneven in run and smudgy.  Check the attached picture for a RNA gel 1.2%. we do it all the time in lab so its pretty standard. You can try the expts you have mentioned with this quality and see if it works.

[ram]

Thank you for the paper p!
@ lihkin Thanks for the comments; I didn't see any attached picture!