Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo

miRNA spike-in control


  • Please log in to reply
6 replies to this topic

#1 comp3v

comp3v

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 11 August 2010 - 01:58 AM

After reading many papers, I arrived to a conclusion that spike-in control is really necessary on extraction of miRNA in some cases (for ex., from plasma). People say that they use synthetic non-related miRNAs (say, C.elegans in case of human samples) but usually don't give a reference of supplier. So, can anybody give a reference to pre-designed synthetic miRNA for using as a spike-in control?
Another way is to order the synthesis of such miRNA from arbitrary supplier, but unfortunately I am not really sure how to design it to make adequate control. I.e., should I just take the full length of mature sequence? or how many nucleotides? without adding anything on 3'/5'? without complement chain?
I would be grateful for any advice. Thanks in advance.

#2 Fizban

Fizban

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 63 posts
0
Neutral

Posted 19 August 2010 - 02:22 AM

After reading many papers, I arrived to a conclusion that spike-in control is really necessary on extraction of miRNA in some cases (for ex., from plasma). People say that they use synthetic non-related miRNAs (say, C.elegans in case of human samples) but usually don't give a reference of supplier. So, can anybody give a reference to pre-designed synthetic miRNA for using as a spike-in control?
Another way is to order the synthesis of such miRNA from arbitrary supplier, but unfortunately I am not really sure how to design it to make adequate control. I.e., should I just take the full length of mature sequence? or how many nucleotides? without adding anything on 3'/5'? without complement chain?
I would be grateful for any advice. Thanks in advance.


The spike in issue is somewhat controversial to me. What i mean is that doing spik-in is sometimes indicated as a control and even used as a normalizer. I'm not so comfortable with this kind of thinking. in a practical way it must be said that it just gives you a control of extraction but only of the synthetic miRNA extraction. in addition just take into account that it 's not usable for normalization because you add a fixed amount of exo miRNA to your sample and different samples have variable RNA content. in conclusion my opinion is that spike in is just useless. In theory one should also note that plasmatic miRNAs could have different sources (exosome/microparticles/protein bound) while the spiked in is just free RNA. it could mean some differences in yield.
by the way if you want to use a spike in just take the complete mature sequence and and just ask your supplier to make it (RNA i would say but DNA works just the same, personal observation).
BE CAREFUL: once you have a synthetic miRNA in your lab you'll be prone to SEVERE contamination of your samples. you'll want to handle it VERY carefully
bye
Fiz

#3 justme

justme

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 20 August 2010 - 07:20 AM

Hello Fizban,
I agree spike in only gives control of extraction, but in this case what do you recommand for qPCR normalization?
I think some used miR-16 or others, but because it is present in red cells, I'm not sure it is a good one. Its expression could vary notably because of hemolysis, which is difficult to control. Or could we add spike in just before retrotranscription starting from same totalRNA quantity ?
Regards,

#4 comp3v

comp3v

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 23 August 2010 - 04:49 AM

Thanks for the answer!

The spike in issue is somewhat controversial to me. What i mean is that doing spik-in is sometimes indicated as a control and even used as a normalizer. I'm not so comfortable with this kind of thinking. in a practical way it must be said that it just gives you a control of extraction but only of the synthetic miRNA extraction. in addition just take into account that it 's not usable for normalization because you add a fixed amount of exo miRNA to your sample and different samples have variable RNA content. in conclusion my opinion is that spike in is just useless.

Indeed, I was mainly speaking about control of extraction, especially considering that efficiency of extraction from plasma can vary significantly (due to different level of lipids/proteins etc). At the same time, it is known that in many pathologies total microRNA content in plasma can be changed drastically - i.e. it is worth to estimate the amount of microRNA and not only relative expression of some miRNA species. And of course, the question of choosing the right normalization control is still open: I could not find any final conclusion for reference microRNA in case of plasma microRNA.


by the way if you want to use a spike in just take the complete mature sequence and and just ask your supplier to make it (RNA i would say but DNA works just the same, personal observation).

yes, that was my question - if I can just take mature sequence. At the same time, could you explain more about using DNA? I thought that if I perform normal RNA extraction, I should selectively extract only RNA and not DNA, and thus DNA oligo cannot be correct control of RNA extraction - or I am wrong here?



With best regards,
V.

#5 Fizban

Fizban

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 63 posts
0
Neutral

Posted 23 August 2010 - 10:43 PM

Hello Fizban,
I agree spike in only gives control of extraction, but in this case what do you recommand for qPCR normalization?
I think some used miR-16 or others, but because it is present in red cells, I'm not sure it is a good one. Its expression could vary notably because of hemolysis, which is difficult to control. Or could we add spike in just before retrotranscription starting from same totalRNA quantity ?
Regards,


there's no such thing as a "global normalizer" so no, you can't use miR 16 just because others did use it (some without even explaining why, but this is a whole different story).
My opinion (personal) is that there are only 3 ways: 1) you are able to quantitate you RNA (difficoult when working with plasma, probably feasible with a bioanalyzer 2100) 2) deep sequencing, you have numbers of reads, it is quantitative for definition, but expensive as far as i know (anyone with cheap deep sequencing available out there? :D ) 3) screening. you measure a lot or all miRNAs in each sample (or a good n) and see which are the most stable. not so subtle and neat but sometimes you have to make it work.

i'm open to new suggestions/ alternative ways/ criticisms
Fiz

#6 Fizban

Fizban

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 63 posts
0
Neutral

Posted 23 August 2010 - 10:52 PM

Thanks for the answer!

The spike in issue is somewhat controversial to me. What i mean is that doing spik-in is sometimes indicated as a control and even used as a normalizer. I'm not so comfortable with this kind of thinking. in a practical way it must be said that it just gives you a control of extraction but only of the synthetic miRNA extraction. in addition just take into account that it 's not usable for normalization because you add a fixed amount of exo miRNA to your sample and different samples have variable RNA content. in conclusion my opinion is that spike in is just useless.

Indeed, I was mainly speaking about control of extraction, especially considering that efficiency of extraction from plasma can vary significantly (due to different level of lipids/proteins etc). At the same time, it is known that in many pathologies total microRNA content in plasma can be changed drastically - i.e. it is worth to estimate the amount of microRNA and not only relative expression of some miRNA species. And of course, the question of choosing the right normalization control is still open: I could not find any final conclusion for reference microRNA in case of plasma microRNA.

What i think is that, as with gene expression, there is no global normalizer (as i said before). each condition asks for it's normalizer and that's simple truth. we can choose to ignore it and treat miR-16 or U6 as "Actin 2.0" but we all know that each miR, like each gene, has its function and is regulated by different conditions. thus, new condition, new normalizer (or old normalizer to be validated).


by the way if you want to use a spike in just take the complete mature sequence and and just ask your supplier to make it (RNA i would say but DNA works just the same, personal observation).

yes, that was my question - if I can just take mature sequence. At the same time, could you explain more about using DNA? I thought that if I perform normal RNA extraction, I should selectively extract only RNA and not DNA, and thus DNA oligo cannot be correct control of RNA extraction - or I am wrong here?

With best regards,
V.


Yep, sorry. i was just thinking about DNA because when i bought synthetic miRNAs 1st time they sent me DNA miRNAs instead of RNA. they work just the same with RT and qPCR when used as a free substrate but of course you have to make RNA if you are extracting that nucleic acid, of course!!!! :P

bye Fiz

#7 Functional Screens

Functional Screens

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 118 posts
4
Neutral

Posted 28 August 2010 - 09:54 AM

For human samples, we used U47 small RNA as endogenous control.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.