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How to increase A260/280 and A260/230 ratio for microarray experiment?


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8 replies to this topic

#1 wntiong

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Posted 04 May 2009 - 04:08 PM

Hi,

I often get a poor ratio of A260/280 and A260/230 ratio from several rna isolations i did.

Since i going to do microarray work, can give me some advise how to increase these ratios? Any tip?

Thank you.

#2 Clare

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Posted 05 May 2009 - 01:43 AM

Hey there :P

Have you tried precipitating your RNA? We have to do this for our DNA as after amplification the ratios are a bit rubbish. After precipitation it's all good ;)

Clare

Hi,

I often get a poor ratio of A260/280 and A260/230 ratio from several rna isolations i did.

Since i going to do microarray work, can give me some advise how to increase these ratios? Any tip?

Thank you.



#3 wntiong

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Posted 06 May 2009 - 12:50 AM

Hey there :wacko:

Have you tried precipitating your RNA? We have to do this for our DNA as after amplification the ratios are a bit rubbish. After precipitation it's all good :D

Clare

Hi,

I often get a poor ratio of A260/280 and A260/230 ratio from several rna isolations i did.

Since i going to do microarray work, can give me some advise how to increase these ratios? Any tip?

Thank you.


can i Re-precipitate the RNA with Isopropanol again or perform more ethanol wash? thanks

#4 Clare

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Posted 06 May 2009 - 05:18 AM

can i Re-precipitate the RNA with Isopropanol again or perform more ethanol wash? thanks
[/quote]

For both RNA and DNA I add 2vol Abs. EtOH and NaOAc to a final conc. of 0.3M. Chuck it in -80 (either ON or at least 30 mins) or put tubes on dry ice for 5-10 mins.
Spin 30 mins, max speed (if using eppendorf tubes).
Remove SN and add 70% EtOH to wash.
Air dry pellet and resuspend in water :wacko:

Clare

#5 wntiong

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Posted 06 May 2009 - 06:33 PM

Thanks for your reply.

Because i am dealing with rna isolation from whole blood, and often get a pink aqueous phase during rna isolation, i am not sure if this affect the rna quality.. any advise? thanks

wenni

#6 Clare

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Posted 07 May 2009 - 12:53 AM

Thanks for your reply.

Because i am dealing with rna isolation from whole blood, and often get a pink aqueous phase during rna isolation, i am not sure if this affect the rna quality.. any advise? thanks

wenni


Do you remove the RBCs before you try and isolate RNA?
C

#7 Vini

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Posted 08 May 2009 - 07:29 AM

Hi,

I often get a poor ratio of A260/280 and A260/230 ratio from several rna isolations i did.

Since i going to do microarray work, can give me some advise how to increase these ratios? Any tip?

Thank you.



Are you getting poor ratios even after purifying your RNA with a purification kit, like that from QIAgen??? because, i usually get much improved ratios after this purification step.

#8 Ahrenhase

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Posted 08 May 2009 - 03:28 PM

Hi,

I often get a poor ratio of A260/280 and A260/230 ratio from several rna isolations i did.

Since i going to do microarray work, can give me some advise how to increase these ratios? Any tip?

Thank you.



Are you getting poor ratios even after purifying your RNA with a purification kit, like that from QIAgen??? because, i usually get much improved ratios after this purification step.


yeah run it through a qiagen RNeasy kit

#9 wntiong

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Posted 11 May 2009 - 04:53 PM

Thanks for your reply.

Because i am dealing with rna isolation from whole blood, and often get a pink aqueous phase during rna isolation, i am not sure if this affect the rna quality.. any advise? thanks

wenni


Do you remove the RBCs before you try and isolate RNA?
C



Hi Clare,

I didnt remove RBC, but stabilize in tri-reagent immediately after blood was drawn.




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