I have isolated RNA from fish retina using Tri reagent. After adding isopropanol, a black coloured RNA pellet has settled down. What is the possible reason behind this unusual black coloured pellet? Whether it will affect any downstream application?
Saurav,
are you using pigmented fish? If so, this could be residue from retinal pigment epithelium. Are you also using Linear Acrylamide to precipitate your RNA? This sometimes leaves a blue hue.
A good idea to avoid RPE in your retinal RNA preparations is to spin your tube down (after lysis) for 3 minutes @ 10,000 g. The pigment will settle on the bottom and you can leave it behind. This is considered good practice, as pigment might inhibit enzymatic reactions.
Good luck.
PS: All of this is true for retinal preparations, unless, of course, you want to include RPE - then this is just what you get!
Hello Felix,
Yes I am using pigmented fish retina. No I am not using Linear Acrylamide to precipitate my RNA sample.
I have already spin down my tube after lysis for 10 mins @ 12,000 g. Pigment already settle down on the bottom after that I have used chloroform for phase separation this step is also ok. I have taken clear aqueous phase then when I put isopropanol It will be appear slight blackish colour. Afterthat centrifuge 10mins @ 12000 g I am getting this black colour RNA pellet.
Is there any process that this pigmented epithelium layer I have to remove properly before homoginization? May be this pigmented layer react with isopropanol. Kindly tell me any reason behind this.
Thanks for your good suggestion.
Hi Saurav,
Seems like you are doing everything right. If this happens only after adding the Isopropanol, I can only think of it being contaminated - what kind of Isopropanol are you using? Is it molecular grade? We use Sigma I9516 and we never had this problem.
Hello Felix,
Isopropanol is not contaminated because I am using same isopropanol for other fish but I got good result as well as I didn't get any black coloured RNA pellet. I am using Sigma molecular graded Isopropanol.
I have already 6-7 times homoginized the retina samples but I was facing same problem everytime. I dont know how to remove this pigmented epithelial layer from retina. Kindly give me any suggestion.
Thanks.
Normally the pigment should settle on the bottom of the tube after spinning it down... but it seems like you are doing that. I would not know why you are getting the black pellet with only one sample as opposed to other samples. Do you have access to an RNA cleanup kit, like Qiagen's RNeasy Mini spin columns? I might be worth it to try cleaning up the reaction once more. Taking a step back, how do you homogenize your sample?
I have homogenized my sample using tissue lyser Qiagen, and trizol method. I am really hopeless and can't find any possible reason behind this one.
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