Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > PCR - Polymerase Chain Reaction Forum > Real-Time PCR and Quantitative PCR Forum
Register Search Today's Posts Mark Forums Read

Real-Time PCR and Quantitative PCR Forum Real-Time PCR and Quantitative PCR Forum


expression levels of different genes within the same sample

expression levels of different genes within the same sample - Real-Time PCR and Quantitative PCR Forum

expression levels of different genes within the same sample - Real-Time PCR and Quantitative PCR Forum


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 02-27-2007, 02:21 PM
Pipette Filler
Points: 1,835, Level: 26 Points: 1,835, Level: 26 Points: 1,835, Level: 26
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2006
Posts: 16
Thanks: 0
Thanked 0 Times in 0 Posts
Question expression levels of different genes within the same sample



Hallo!

Does anyone has a clue if it is possible and how to compare expression levels of several different genes within the same sample, by real-time RT-PCR?

Thanks,

stef
Reply With Quote
  #2  
Old 03-24-2007, 04:08 PM
Pipette Filler
Points: 1,355, Level: 21 Points: 1,355, Level: 21 Points: 1,355, Level: 21
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2007
Posts: 9
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: expression levels of different genes within the same sample

Sure,

there are two ways:

1. You can run SYBR green real-time PCR reactions with 1 ul sample each. The amount of genes you can analyze depends on the amount of sample. But you always have to run housekeeping genes so that you can account for your pipetting error. So each gene run in triplicates = 3 ul plus your housekeeping genes. Let's say you want to analyze 10 genes and 2 housekeeping genes = 12 genes total x 3 ul each = 36 ul sample +/- 5 ul to be on the safe side, so you need ca. 40 ul. But remember that you do not need high concentration DNA samples for real-time PCR.

2. Multiplex real-time PCR reactions. These are based on individual primers and gene-specific taq-man probes. The taq-man probes or molecular beacons are tagged with different fluorescent dyes to the machine can distingiush them when measuring the fluorescence. The number of genes to be analyzed depends on your real-time PCR machine. Some can do only one color at a time, most of them do up to 5 colors. So that means you can (theoretically) run 5 genes in 1(!!!) reaction. However, it is difficult to set up because you need to make sure that the individual primers do not amplify more than your desired gene. The more genes you want to analyze, the more difficult it gets to optimize. So this second way is only worth the work if you are limited on DNA sample.

Let me know when you have more questions!!!
Reply With Quote
  #3  
Old 05-18-2007, 06:24 AM
Pipette Filler
Points: 1,618, Level: 24 Points: 1,618, Level: 24 Points: 1,618, Level: 24
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2006
Posts: 14
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: expression levels of different genes within the same sample

and if you need some software tools, there are many out there for designing real time PCR primers and probes such as Primer3 or Beacon Designer
Reply With Quote
  #4  
Old 05-22-2007, 05:26 PM
Pipette Filler
Points: 1,835, Level: 26 Points: 1,835, Level: 26 Points: 1,835, Level: 26
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2006
Posts: 16
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: expression levels of different genes within the same sample

hi PeterScherp

thanks for your time to write this detailed instructions!

unfortunatelly, I have a feeling that I did not explain well my problem/question.

so, usually you want to compare expression levels of desired gene(s) between two sets of samples (control and treated, cell/tissue-1 and cell/tissue-2, or so on...). it is clear to me how to do that.

what I do not know how to do is to compare expression levels of gene A and gene B (and C, D, E...) within the _same sample_, so to be able to answer if A is expressed at higher levels than B (and so on...). absolute quantification would give me desired answer (i.e. x moleculs of A, y moleculs of B), but I am not sure how/if I could obtain this answer if not using standard curve with apsolute concentrations (from plasmid, for example).

so far, I came to conclussion that this would not be possible to do (or would be too demanding) with SyberGreen technology since ct-s depend on the length of Pcr products, among other things. maybe with TaqMan it would be feasable, but I did not encounter relevant papers or software for that kind of analysis.

hope I made myself more clear this time.

thank you anyway,

stef
Reply With Quote
Reply

Tags
expression , genes , levels , sample


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Human Cytome Project - Update 24 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 1 08-01-2010 02:18 PM
Human Cytome Project - an idea - Update 19 April 2005 Peter Van Osta Cell Biology and Cell Culture 1 06-01-2009 02:17 PM
qPCR NEWSLETTER - October 2006 Editor www.Gene-Quantification.info Protocols and Methods Forum 0 10-26-2006 12:25 PM
A Human Cytome Project - an idea - Update 14 March 2005 Peter Van Osta Cell Biology and Cell Culture 0 03-14-2005 02:27 PM
Human Cytome Project - Update 6 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 0 01-06-2005 11:18 AM


All times are GMT. The time now is 10:21 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.18677 seconds with 16 queries