RNA extraction/electrophoresis problem - (Oct/11/2007 )
Hi, I am facing a weird problem with my RNA sample (run on undenatured agarose gel). The RNA forms a fireball instead of a band and is located below the loading dye band (bromophenol blue , approximately 2kb).
On formaldehyde gel, the same thing happens. (Followed QIAGEN Plant RNeasy mini kit).
My RNA was extracted from roots using manual methods. Liquid nitrogen was used to freeze the roots immediately after being removed from culture. Cell distruption acheived via pestle and mortar grinding. All material were DEPC treated. I have tried using kits to extract my RNA but they don't work for me.
Is the RNA degraded ? Is there protein contamination ? Any suggestion on fixing this ?
Before this I used to get ok RNA but suddenly my extractions gives me this kind of results. I checked my solutions by extracting RNA from leaves and they work so I don't think my solutions have RNAse contamination. Help please.
Please help! Thank you.
sorry, dont wanna disappoint you but your RNA is degraded... possibly occuring prior to lq N2? sometimes when you change all the buffers etc, rule out RNAse then it comes down to your initial RNA preservation. Maybe you aren't grinding up and getting into lysis (presumably some GITC-phenol solution?) fast enough. Are you taking larger samples or something else different from previously? Wish I had better news. HTH and good luck!
thanks for your reply. so that is a problem of RNA degradation is it? at first i thought RNA degradation will result in smearing on agarose gel. someone told me that the sample which run faster than the dye is probably protein but i doubt it is true.
i am extremely sure that i grind the root to become very fine powder but still i couldn't get result. before this, i didn't grind as fine as the later but surprisingly i can get RNA although the yield is quite low. yes i am using larger sample than before. last time i used 2.5g of sample per tube but since the yield is quite low so i increased it to 5g per tube. of course this takes me quite some time to grind the sample into fine powder, i think around 20 minutes. but what i think is as long as i didn't let the sample to thaw, it is still fine, isn't it? what is GTIC-phenol? i am currently using non-euilibrated phenol. i just found out from this forum that RNA extraction favor acid-phenol so i had change the phenol from pH 8 to pH 4.7, as suggested by one of the forum member here. but still it couldn't work.
ok, now comes to your question. yes, i did change something from my previous work. for your information, i am dealing with in vitro plantlet. before i face this problem, i used to culture my plantlet in solid medium and i harvest the root on my work bench, meaning it is not in a sterile condition. however, it took a very long time grow in solid medium, approximately 2-3 months. besides, once i open up the culture flask outside a laminar hood, i can't use the shoot for subculture anymore. so i try to culture my plantlet in liquid medium and it works perfectly well where it needs 3-4 weeks to grow. then, in order to use the shoot for subculture purpose, i harvest the roots in a laminar hood. i admit that it took quite a long time for me to harvest the root. so after i separate the roots from the plantlet, immediately i put it back into the medium. i think this is the time when RNA start to degrade. therefore, i change my method of harvesting again. this time once i separate the root from the plantlet, immediately i dipped it in liquid nitrogen. this is what i did for every planlet until all the planlet finish. but still, i don't get any result.
i have try to search for some reagents that will help to protect RNA from degradation after harvesting, that is RNAlater RNA Stabilization Reagent from QIAGEN but it can be apply for animal tissues only. I found another one from Ambion, it is Plant RNA Isolation Aid but i don't think can inhibit RNase activity. it is just a reagent like Trizol.
or could it be possible that the RNA content will lost due to different usage of culture medium?
rna degradation may be due to too high sample/extraction buffer ratio. I would first try to use smaller samples, even less than you used when you were growing your plantlets on solid medium. Perhaps the faster growing plantlets on liquid medium have a higher content of endogenous RNAses. And it is not true that you have to use acidic phenol, pH 8 buffered phenol is all right as well.
thank you andrea massimo. may i know what is the ratio of sample/extraction buffer that you normally use? i am currently using 1:10, meaning 1g of sample plus 10ml of extraction buffer.
I use 1:5 to 1:10 (1:5 for samples with low rna content, 1:10 for rich sources as young leaves). Since you use 1:10, that seems to be all right. Just also make sure to have space enough in the tubes for proper mixing. I usually don't fill the tubes more than a half. After grinding, samples should be quickly and thoroughly mixed with sample buffer, avoiding clumps. They should be finely dispersed in the buffer during the initial steps of the extraction. If also this point is all right, you may really need to look for another method. I've got little experience with roots, but the CTAB method performed well in some cases. You can find references about it in a recent topic about rna extraction from flowers.
i think this time i want to try the 1:5 ratio because usually when i use 1:10, the tube is almost full and maybe the improper mixing might cause the lost of RNA. another problem i am facing now is that we don't have homogenizer in our lab. so after i grind the roots, immediately i pour the pre-cooled extraction buffer into the mortar. but then the mixture of extraction buffer and sample powder will be frozen and it become hard to homogenize with pestle. how do you usually overcome this problem?
1) have ready in liquid nitrogen (N2) the empty tubes (capped) you are going to use
2) grind a sample, remove one tube from N2 and transfer the sample in it, with a spatula chilled in N2, cap tight and return the tube with the powder in N2, proceed in this way till the last sample.
3) remove a tube with the ground sample from N2, pour the buffer in the tube, SWIRL BY HAND AND WAIT UNTIL ALL THE RESIDUAL LIQUID NITROGEN HAS EVAPORATED BEFORE CAPPING (otherwise the tube may explode), cap tight and vortex well. Proceed in this way for all samples.