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

The reliable data of microRNA expression from SYBR-stem loop PCR or ABI Taqman m


  • Please log in to reply
8 replies to this topic

#1 ljchen

ljchen

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 16 January 2010 - 02:04 AM

Hi, dear all:

Recent, I do the microRNA expression. First, I used the ABI Taqman microRNA array to screen my RNA sample and got the microRNA prolife. Then, I used the SYBR stem-loop PCR system (the primers designed by myself) to check the data from the ABI array. I am curious about the accuracy of data. Does anyone think about this question?? Because I have gotten data from ABI array, but some of them can't be expressed the same data in SYBR stem-loop PCR system, particularly the ratio of microRNA expression. The expression patten are same in both system, however, the ratio is dramatically difference. Seems like the ratio is huge in the ABI microRNA array. I don't know I sould trust which one. Does anyone have same question with me??

Thank you!!

#2 Fizban

Fizban

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 63 posts
0
Neutral

Posted 18 January 2010 - 12:36 AM

Hi, dear all:

Recent, I do the microRNA expression. First, I used the ABI Taqman microRNA array to screen my RNA sample and got the microRNA prolife. Then, I used the SYBR stem-loop PCR system (the primers designed by myself) to check the data from the ABI array. I am curious about the accuracy of data. Does anyone think about this question?? Because I have gotten data from ABI array, but some of them can't be expressed the same data in SYBR stem-loop PCR system, particularly the ratio of microRNA expression. The expression patten are same in both system, however, the ratio is dramatically difference. Seems like the ratio is huge in the ABI microRNA array. I don't know I sould trust which one. Does anyone have same question with me??

Thank you!!


generally speaking, do NOT trust results from ABI arrays as standalone results, in particular if u r doing pre amplification. u have data that must be verified for each mirna of interest. these arrays are good, quick and sensitive but not reliable for Cts and false negative.
Fiz

#3 rnarm

rnarm

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 18 January 2010 - 03:00 AM

A word of advice from myself is to discount anything over 35 cycles on the array because speaking with ABI they suggested anything over 35 is not expressed. For my data, I chose a 35 CT as my cuttoff for undetermined, not 40 CT.

I also find the CTs for endo and targets come up later in single assays compared to TLDAs (Im using taqman validation). So a gene which might come up at 30 CT in a TLDA might come up at 35 CT in single assays when using 1 ug total RNA in TLDAs (endo also increases). So select your genes for validation carefully from your TLDAs.

#4 ljchen

ljchen

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 24 January 2010 - 10:04 PM

Hi, dear all:

Recent, I do the microRNA expression. First, I used the ABI Taqman microRNA array to screen my RNA sample and got the microRNA prolife. Then, I used the SYBR stem-loop PCR system (the primers designed by myself) to check the data from the ABI array. I am curious about the accuracy of data. Does anyone think about this question?? Because I have gotten data from ABI array, but some of them can't be expressed the same data in SYBR stem-loop PCR system, particularly the ratio of microRNA expression. The expression patten are same in both system, however, the ratio is dramatically difference. Seems like the ratio is huge in the ABI microRNA array. I don't know I sould trust which one. Does anyone have same question with me??

Thank you!!


generally speaking, do NOT trust results from ABI arrays as standalone results, in particular if u r doing pre amplification. u have data that must be verified for each mirna of interest. these arrays are good, quick and sensitive but not reliable for Cts and false negative.
Fiz


Thank you for your description, Fiz.

#5 ljchen

ljchen

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 24 January 2010 - 10:13 PM

A word of advice from myself is to discount anything over 35 cycles on the array because speaking with ABI they suggested anything over 35 is not expressed. For my data, I chose a 35 CT as my cuttoff for undetermined, not 40 CT.

I also find the CTs for endo and targets come up later in single assays compared to TLDAs (Im using taqman validation). So a gene which might come up at 30 CT in a TLDA might come up at 35 CT in single assays when using 1 ug total RNA in TLDAs (endo also increases). So select your genes for validation carefully from your TLDAs.


Dear rnarm:

Thank you for your suggestion. I don't understand what do your mean about the "TLDA"?? What's this?

Recent, I gradually realize it is very diffical to find out a microRNA of interest. However, I have more confidence to trust my data from SYBR stem-loop. How about the Northern blot?? Does it trustworthier than qPCR?

#6 Fizban

Fizban

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 63 posts
0
Neutral

Posted 25 January 2010 - 05:15 AM

A word of advice from myself is to discount anything over 35 cycles on the array because speaking with ABI they suggested anything over 35 is not expressed. For my data, I chose a 35 CT as my cuttoff for undetermined, not 40 CT.

I also find the CTs for endo and targets come up later in single assays compared to TLDAs (Im using taqman validation). So a gene which might come up at 30 CT in a TLDA might come up at 35 CT in single assays when using 1 ug total RNA in TLDAs (endo also increases). So select your genes for validation carefully from your TLDAs.


Dear rnarm:

Thank you for your suggestion. I don't understand what do your mean about the "TLDA"?? What's this?

Recent, I gradually realize it is very diffical to find out a microRNA of interest. However, I have more confidence to trust my data from SYBR stem-loop. How about the Northern blot?? Does it trustworthier than qPCR?


TLDA stands Taqman Low Density Arrays (ABI cards for miRNA screening).
northern blot can be useful if u have:
1) a LOT of RNA
2) a miRNA which is expressed enough to be detected (which is not always the case, but u would discover it only AFTER your northrn is done)

my suggestion is stick to the qPCR method you find more reliable, there's a lot of work to be done from screening to real results!!! ;)
fiz

#7 shadow

shadow

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 03 March 2010 - 09:01 AM

A word of advice from myself is to discount anything over 35 cycles on the array because speaking with ABI they suggested anything over 35 is not expressed. For my data, I chose a 35 CT as my cuttoff for undetermined, not 40 CT.

I also find the CTs for endo and targets come up later in single assays compared to TLDAs (Im using taqman validation). So a gene which might come up at 30 CT in a TLDA might come up at 35 CT in single assays when using 1 ug total RNA in TLDAs (endo also increases). So select your genes for validation carefully from your TLDAs.

When I use Sybr-Stem loop way to detect my microRNA, even the NTC got a Ct value around 30. I know it's false positive, but I don't know how to get rid of it. The upl probe method showed no signal for the NTC.

#8 CamillaSteenJensen

CamillaSteenJensen

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 13 December 2011 - 02:13 AM

I have the same problem with my NTC. It comes up around ct 28, and I cant seem to get rid of this false postiiv result. I've tryed alomost everything, but nothing seems to been working :-(

#9 Fizban

Fizban

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 63 posts
0
Neutral

Posted 15 December 2011 - 02:36 AM

Hi all,
i just came back on this topic and have an attentive read. i'd advice to use commercial primers and probes for miRNA qPCR, they're more reliable than hand made. if u have problems with NTC results try taqman probes instead of SYBR, less false positives (in single assays) and no primer dimer signals.
hope it helps
Fiz




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

©1999-2013 Protocol Online, All rights reserved.