I am working on human tissue samples. I am studying expression of some genes. For that I have to make cDNA from RNA which I isolate from tissue samples.
We can use various methods like chopping, Liquid nitrogen for RNA isolation.
Right now I am using chopping and mixing method for RNA isolation from tissue samples. I am getting a good concetration of RNA but when I checked RNA integrity on gel I got a smeared pattern instead of 2 clear bands. What precaution should I take to get intact RNA? Is there any alternative method I can use for this.
Use the autoclaved equipments after RNAse inhibitor treatment(eg. DEPC) for extraction; The buffer should be prepared in a same way or else try with any kit like TRI reagent from sigma chemicals.
good luck!!! Enjoy the science of RNA
I think the first thing to check is that you are running the gel properly. Are you using fresh buffer and only running for 10-15 minutes? If you have access to a Bioanalyzer then try running some of your samples through that to double check if your RNA integrity is actually at fault.
The tips I have for RNA extraction is to work quickly, put samples as soon as possible into solutions which kill/inhibit RNases (normally the solutions in which you mince/homogonize, eg Trizol), and keep things cold.
it seems your RNA has degraded you can try these precaution,
First clean with detergent and wipe with alcohol then bake all the equipment you use for RNA isolation i.e. mortar pestle, spatula, etc. in 100 degree for overnight..
prepare 1% DEPC and keep for mixing in magnetic stirrer for overnight then autoclave it..
wipe out the place which you are going to use for RNA isolation with 70% ethanol and DEPC..
You can use the qiagen RNA extraction kit for RNA isolation.it gives you the high quality of RNA from tissue samples...
GOOD luck.
Cleaning all equipment as suggested above is important.
Chopping and mixing may not be enough for a complete homogenization depending on the tissue.
A complete homogenization is required to make sure that your RNA extraction buffer/reagent penetrate all the tissue and inactivate RNases.
And what is important, keep your samples on ice during the whole procedure of RNA isolation.
I have replaced grinding in liquid nitrogen with homogenizing in tri-reagent (trizol) using a Qiagen Tissuelyzer (sp.?). The maching uses a stainless steel bead added to your sample to beat the tissue into solution. We use this method on adipose tissue and have pretty good success. After the trizol prep we further clean the RNA with a GE RNA miniprep kit (same as the Machery-Nagel kit). The process is somewhat tedious but we get very clean, intact RNA for both qPCR and cloning purposes.
I think Anil has a point here. Most of us take all precautions during RNA isolation but sometimes we overlook the electrophoresis part. I hope your electrophoresis unit is RNase free and you prepare your gel in RNase free buffer. Its always better to denature your RNA at 65 deg C for 5 min and placing on ice for 2 mins before loading your gel.
Not using Ice cold ethanol for washing might also lead to such results.
Good Luck.
If you use the same gel electrophoresis system for running DNA samples, you must clean it before use it to RNA , so I suggest to clean it with H2O2, and use new and autoclave tips, solutions also autoclave equipments to prevent any RNAse inhibitor.
RNA Express reagent from HI-media is also giving good yields... I use it routinely for RNA Isolation... you just need to have the DEPC treated materials and A separate laminar hood ...
homogenizer is a better option than nitrogen and mortar and pestle treatment. In addition to this precautions with RNAse as mentioned in other answers will difinitely give you very good quality RNA with excellent RIN Scores.
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