Not too sure what you mean by your question? Is it for prokaryotes or eukaryotes?

Care to provide your current protocol for people to discuss?

Thanks

i am sorry for incorrect sentence. working on eukaryotes.

i am not getting 28s r RNA double of 18s r RNA intensity (as in standard condition ) in my RNA isolation although bands are sharp.

using TRIZOL based isolation method .

The intensity between 28S and 18S is only a relative guide for comparison. A long as they are equally intense and no high degree of smearing, the quality shouldn't be too bad.

Cells at different stages of their cycle can have different rRNA expression too, check your samples aren't near the end of life cycle.

If not, simple lab practice to minimise RNase contamination should be pretty easy to find online. Many companies (Ambion, Qiagen, NEB...etc) provide decent guidelines for RNase troubleshooting

i need to correlate my gel image(RNA) with RIN number from bioanalyser. i also want to correlate 260/280 ratio 260/230 ratio with bioanalser RIN number ? i will also attach my gel image for your kind consideration. because it is not possible to run every sample on bioanalyser for RNA quality measure.

Trizol is actually great for RNA integrity. I used to get RIN around 8ish, which is sufficient for cDNA library prep. But my PI wanted me to really get a perfect 10 so we don't get "garbage in, garbage out". So I changed a few things.

1. Cleaned my bench before begin. Wipe down everything. Use filtered pipette tips. 

2. Purchase commercial RNAse free/DEPC H2O and use it to make your 75% EtOH.

3. Purchase your 3M NaOAC. 

4. Just minimize the amount of hand waving and use absolute sterile practices.

5. Don't try to get that last 10ul when you do your chloroform separation to avoid contamination, quality over quantity.

You could make all those reagents needed for RNA isolation, but make sure they are clean. Change out all the old reagents when you do your next try since you probably opened and closed them so many times. The reason we get commercial is that we can absolutely sure they are nuclease free. Everything became 10 and PI is happy.

hi jeff li-

i am also looking for RNA later , if i can purchase , in that case i am worried about storage because what i want is just to store my tissue in RNA later and then directly to -80 up to trizol step. and rest you said is reasonable to follow up. 

i am not sure that i can go for RNA later in such a manner?

I think it should work. Because RNAlater just help you preserve tissue and RNA without the hassel of freezing samples. So, from what I have read, you aspirate out the RNAlater, and process with trizol downstream. RNAlater should not interfere with Trizol based on what invitrogen said.

However, I have never used RNAlater myself, I have always directly processed with Trizol, it's the safest way. 

Goodluck.