I want to isolate RNA from testicular tissue using Trizol. Everytime when I run the gel after isolating RNA, the bands are getting smeared. All the precautions have been taken care. All the solutions are treated with DEPC, glasswares and plasticwares were made nuclease free & autoclaved. We think the problem is occurring in the first step of tissue homogenisation. Also the testicular tissue was 4 days old during homogenisation. We are using a micropestle for homogenising the tissue in the 2ml eppendroff tube. Is this right or we should follow some other method for homogenising? can we use Liquid Nitrogen?
Can anyone suggest me a better way?
"Also the testicular tissue was 4 days old during homogenisation"
Was your tissue soaked in RNALater or any other protective reagent when the tissue was gathered? Tissue needs to be soaked in a protective solution such as RNALater in order to preserve it for analysis, When your sample was stored 4 days, how was it stored? You should always be storing samples for RNA in -80*C. Smeared bands usually indicate RNA degredation. If you were having extraction issues, you should be getting low RNA yield.
You should grab an RNA sample from collegue that is confirmed to be intact and run it on your gel with your samples. This can serve as a positive control. If you run your samples with a positive control, and you still get smearing on your sample only but not pos control, then it is likely that your samples are degraded.
http://image.slidesharecdn.com/rnaextraction-121230121003-phpapp02/95/rna-extraction-22-638.jpg?cb=1357578125
Hello Gopalakrishnan Chandrasekaran,
I have used liquid nitrogen for homogenization of bone tissue to isolate the RNA. I dont have problem with the smear.
Regardless, without proper storage and preservation, RNA begins to degrade within hours
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056507 (figure 1)
Imediately snap frozen in liquid nitrogen, and then disruption with a cold mortar an pestle with more liquid nitrogen may help you. Be shure to do not use more tissue than ten percent mass/volume with Trizol.
Also do liver as positive control o the process.
Like many respondents, I also suggest "snap freeze tissue in Liquid nitrogen, pulverise in a mortar and pestle under more liquid nitrogen" (I suggest you place the mortar in a bed of dry ice to keep it cold, too).
Add the trizol directly to the frozen powder.
I would further add: testis tissue can be very fatty, and fatty tissue is (in my experience) more problematic for RNA extraction. My advice would be "use a lot of TRIzol". If you'd usually use 1ml per 100mg of power, double it to 2ml, or even 3ml.
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