Wake7 asked in Science & MathematicsBiology · 1 decade ago

Cause of low 260/230 ratio on Qiagen RNeasy kit for RNA extraction?

Ignore this, it's not a question. It's to help future people are googling this issue. I came across some yahoo answers a few times when I was trying to figure out the problem but no solutions work, so this is for anyone else in the future who has an issue.

First are the things we have heard on the internet and tried:

1. Heat up the RLT buffer to 37C before use. Let it cool a bit but once added to the frozen cell samples, take the samples off ice. The whole point is to stop the RLT from getting too cold and forming crystals.

2. Ensure your gloves aren't covered in white stuff (salty RLT) when you are doing the washes.

Now here is what solved the extremely annoying and expensive issue for us:

when doing the washes (RW1 and RPE) invert the column several times so that the buffers can wash the lid. Then, roll the spin column (like you would roll a barrel) with your fingers to make sure the buffer has really washed the inside of the tube.

This makes sense; the buffer volumes (700ul and 500ul) are not enough to fill the column and reach the lid. If you don't do this, you will notice small white crystals all over the underside of the cartridge lid. This is RLT, and it can come down during the elution. Also, rolling the wash makes it actually wash the cartridge, as opposed to just sitting there. It's possible that this is why you are supposed to squirt the RNAse free water directly onto the filter, but we have not tested this.

here's some keywords so people can find this:

RLT contamination

low 260/230 ratio

bad 260/230 ratio

contaminated RNA

guanidine contamination

Here's an example of everyone disagreeing on the issue:

http://www.protocol-online.org/biology-forums/post...

A ratio of 1.8 or higher is needed for array processing.

Also, something else you might try is sucking the flow through out of the collection tubes with an aspirator instead of just dumping them out (as dumping them out isn't nearly as clean).

Qiagen also recommended letting the RPE and RW1 remain in the cartridge for about 2 minutes so that they can wash. (Doing the inversion and rolling of 16 samples as I recommend above easily takes 2 minutes anyway).

Good luck. Also, if using the qiagen tissue lyser, make sure you know that the farther out the samples are in the blocks, the more they get shaken and the more lysis occurs. (There are 3 rows, use the row farthest away from the lyser)

Update:

No offense but your post is pretty bad... Labs do not have the time or the money to spend getting bad RNA and finally figuring out its because they didnt invert the cartridge...that does not teach them anything about science, it only gets rid of lab money. There is nothing wrong with what I'm doing.

You are also arguing two different things, it's fine that you have a problem with the kits since they teach nothing about isolating RNA...but you should have no problem helping people skip little issues when it comes to using the kit. Telling people to invert and roll the cartridge has nothing special to do with scientific methods for the experiment...

3 Answers

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  • 1 decade ago
    Favorite Answer

    I think most people who uses this kit already knows it ... or let them trouble shoot things .. trouble shooting is the way to learn issues. students in lab now a days have no idea why and how a certain experiment works because of kits like this .... giving them a chance to trouble shoot will give them an opportunity to actually look into the process and methods.

    Source(s): PhD candidate
  • Anonymous
    4 years ago

    Qiagen Rna Extraction

  • byro
    Lv 4
    4 years ago

    acquiring extreme high quality, intact RNA is the 1st and generally the main serious step in appearing many elementary molecular biology experiments, which contain Northern prognosis, nuclease safety assays, RT-PCR, RNA mapping, in vitro translation and cDNA library shape. to prevail, however, the RNA isolation technique might desire to contain some important steps the two in the previous and after the fully RNA purification. in this article discusses various RNA isolation procedures and procedures of increasing RNA yields.

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