1
$\begingroup$

I used Qiagen's RNeasy Mini Kit to isolate RNA from 5*10^5 C28/I2 (immortalized human chondrocytes). However, my yield is low (~25 ng/ul), but my 260/280 ratio is great (~2.3), and my 260/230 ratio is tragic (~0.06). I have already thrown the spin column away, but I would like to know 1) if I could raise my 260/230 ratio for the 40 ul of RNA I extracted, and 2) if I could precipitate the RNA out of solution so as to make a more concentrated solution.

Please let me know if further details are necessary.

Thanks!

$\endgroup$
3
  • 1
    $\begingroup$ You can easily precipitate the RNA using either Ethanol or Isopropanol. I would do a Phenol-Chloroform extraction before that, too. But does this make sense? When you are working with a cell line, I would rather make a fresh prep than wasting time and money into saving the poor quality RNA. $\endgroup$
    – Chris
    Jul 16, 2014 at 22:31
  • $\begingroup$ These cells grow really slowly and I have some simple PCR that I need to run within the next couple days, so I'd ideally like to spare it. I've just never had these issues before. How do I precipitate the RNA using ethanol? $\endgroup$
    – yelx
    Jul 16, 2014 at 22:35
  • $\begingroup$ did you modify the amount of RLT buffer in the Qiagen RNAeasy mini kit? If yes, how much did you use? $\endgroup$ Sep 21, 2022 at 16:12

1 Answer 1

1
$\begingroup$

RNAeasy kit will not usually give phenolic contamination. Your poor 260/230 is perhaps because of low yield; check the absolute absorbance values. Low 260/230 (and low yield) also happens because of improper lysis. I would advise that you do the cell lysis using more effective methods. I would not advise another phenol-chloroform or alcohol precipitation step- you will lose your RNA. With what you have now, run a little bit on the gel to see the integrity. Also try heating the dissolved RNA at 65-70deg for 10-15min and measure OD again (use nanodrop or a similar device)

$\endgroup$
3
  • 1
    $\begingroup$ The low 260/230 ratio is indicative of guanidine contamination I believe. The RNeasy "RLT buffer" contains guanidine to my knowledge. $\endgroup$
    – yelx
    Jul 17, 2014 at 17:05
  • 1
    $\begingroup$ It does. but column based purification generally doesn't leave these contaminants.. $\endgroup$
    – WYSIWYG
    Jul 18, 2014 at 4:16
  • $\begingroup$ How did you lyse them? WYSIWYG is probably onto something here. $\endgroup$ Mar 3, 2019 at 20:48

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .