Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > RNA Techniques Forum
Register Search Today's Posts Mark Forums Read

RNA Techniques Forum Post and discuss RNA methods and RNA science related topics. RNA extraction, cDNA synthesis, RNA EMSA, and other RNA protocols.


Quantifying mRNA levels

Quantifying mRNA levels - RNA Techniques Forum

Quantifying mRNA levels - Post and discuss RNA methods and RNA science related topics. RNA extraction, cDNA synthesis, RNA EMSA, and other RNA protocols.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 02-01-2011, 06:28 PM
Pipette Filler
Points: 689, Level: 13 Points: 689, Level: 13 Points: 689, Level: 13
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2010
Posts: 21
Thanks: 1
Thanked 0 Times in 0 Posts
Default Quantifying mRNA levels



Hi,

I am new to this field. Have not had much experience with RNA work.
Can anyone tell me how does one quantify mRNA ? I am doing induction studies and have to check the mRNA levels and how they differ at differnet induction levels/times
Thank you
Reply With Quote
  #2  
Old 02-01-2011, 07:02 PM
luisillo's Avatar
M.D/Ph.D
Points: 3,877, Level: 41 Points: 3,877, Level: 41 Points: 3,877, Level: 41
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2010
Location: Mexico
Posts: 352
Thanks: 19
Thanked 98 Times in 90 Posts
Default Re: Quantifying mRNA levels

Real-time PCR (qPCR). The technique is similar to a conventional RT-PCR: You isolate your RNA, retrotranscribe it to cDNA and do the PCR. In qPCR case, you need special cyclers which can read the signal of dyes known as reporters. These dyes bind to new DNA strands, so, the higher the signal, the more new DNA is synthesizing, which ultimately mean you have a higher expression of your target mRNA. Of course, the expression of your target mRNA has to be compared with the expression of housekeeping mRNA expression.

Check this link for general info:

[Only registered users see links. ]
Reply With Quote
  #3  
Old 02-04-2011, 04:25 PM
Pipette Filler
Points: 689, Level: 13 Points: 689, Level: 13 Points: 689, Level: 13
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2010
Posts: 21
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re:primer design of qRT-PCR

Thanks. The link was really helpful.
How does one design primers for the q RT-PCR?
I mean how do I start? Sorry for sounding dumb but, I am a novice in this field....
Reply With Quote
  #4  
Old 02-06-2011, 02:24 AM
luisillo's Avatar
M.D/Ph.D
Points: 3,877, Level: 41 Points: 3,877, Level: 41 Points: 3,877, Level: 41
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2010
Location: Mexico
Posts: 352
Thanks: 19
Thanked 98 Times in 90 Posts
Default Re: Quantifying mRNA levels

As far as I know, it's the same as conventional PCR primers, I mean, the same primers can be used for both conventional PCR and for qPCR. As for the designing, you ought to have the sequence of your gene to begin with. Once you have it you can insert it in the Primer design software (i.e. Primer3) and it should give you some suggestions of primer sequences.
Reply With Quote
  #5  
Old 02-06-2011, 02:15 PM
Pipette Filler
Points: 689, Level: 13 Points: 689, Level: 13 Points: 689, Level: 13
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2010
Posts: 21
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re: Quantifying mRNA levels

Thanx. I have the sequence of the enzyme whose mRNA levels need to be quantified at different induction conditions. But, based on other published work; it just not the enzyme itself but, other genes that are involved in its expression.

So my guessing is until I have the whole cassette of all the genes involved in the expression process doing a experiment of such a kind is going to be of little help right?
Reply With Quote
  #6  
Old 02-08-2011, 08:59 PM
luisillo's Avatar
M.D/Ph.D
Points: 3,877, Level: 41 Points: 3,877, Level: 41 Points: 3,877, Level: 41
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2010
Location: Mexico
Posts: 352
Thanks: 19
Thanked 98 Times in 90 Posts
Default Re: Quantifying mRNA levels

It depends. Measuring how the induction affects the enzyme's mRNA expression is important unless it has been already done (I don't know much about the topic). In this case I think it'll make you see if your induction conditions affect or not the expression of the enzyme's mRNA, and depending on your results, you might decide to study or not the expression of those other genes involved in order to know the mechanisms involved.

Considering this, I think the experiment with only the enzyme's mRNA IS important as a starting point, then again, qPCR is kinda expensive. My advice is: if money isn't a problem, then do it. But anyway, that's just my opinion, and as I said before I'm not very knowledgeble on this topic (induction). Guess you better discuss it with your boss first.

The best of luck.
Reply With Quote
The Following User Says Thank You to luisillo For This Useful Post:
admin (02-09-2011)
  #7  
Old 02-09-2011, 08:14 PM
Pipette Filler
Points: 689, Level: 13 Points: 689, Level: 13 Points: 689, Level: 13
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2010
Posts: 21
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re: Quantifying mRNA levels

Thanks! So I can use the primers which I designed to pull out the enzyme from the gene(gDNA) and do a qPCR (With RNA as a template) ??
Reply With Quote
  #8  
Old 02-09-2011, 09:25 PM
luisillo's Avatar
M.D/Ph.D
Points: 3,877, Level: 41 Points: 3,877, Level: 41 Points: 3,877, Level: 41
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2010
Location: Mexico
Posts: 352
Thanks: 19
Thanked 98 Times in 90 Posts
Default Re: Quantifying mRNA levels

That might be possible if your working with prokaryotic organisms. In eukaryotic might be a little difficult because the sequence change: genomic DNA has introns which are absent in a mRNA sequence. When you design your primers you have to put the sequence you want to amplify and as I stated above, it might differ from gDNA to mRNA, which will ultimately alter the sequence of your primers and thus their specificity.

I guess you'll have to figure that question yourself based on your knowledge about the topic.

Again, I wish you luck.
Reply With Quote
The Following 2 Users Say Thank You to luisillo For This Useful Post:
admin (02-09-2011), Sr_08 (02-10-2011)
  #9  
Old 02-10-2011, 10:29 PM
Pipette Filler
Points: 689, Level: 13 Points: 689, Level: 13 Points: 689, Level: 13
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2010
Posts: 21
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re: Quantifying mRNA levels

Thank u for replying to my post! I have really learnt a lot in these few days!!
One more question,
So, cells in uninduced state (will they be the housekeeping mRNA genes?) can be used to compare
expression of the target mRNA ???

Last edited by Sr_08; 02-10-2011 at 10:34 PM.
Reply With Quote
  #10  
Old 02-11-2011, 06:34 PM
luisillo's Avatar
M.D/Ph.D
Points: 3,877, Level: 41 Points: 3,877, Level: 41 Points: 3,877, Level: 41
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2010
Location: Mexico
Posts: 352
Thanks: 19
Thanked 98 Times in 90 Posts
Default Re: Quantifying mRNA levels

Uninduced cells will be the control for the assay. I think it is important that you include that experiment. But, for housekeeping gene I mean a mRNA that is constitutively expressed in the cell, some examples are B-actin mRNA or ribosomal subunits mRNA. By comparing the constitutively expressed mRNA expression against your target mRNA expression you can tell if there are differences in you gene's expression within the same cell culture (or condition or whatever you prefer to call it). In this way you discard random variability between experiments.
Reply With Quote
The Following User Says Thank You to luisillo For This Useful Post:
admin (02-11-2011)
Reply

Tags
levels , mrna , quantifying


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
>>>> LOWER CHOLESTEROL <<<< Janis Stroud Forum Physik 0 09-22-2009 04:39 PM
Suppression of Protein Levels but not mRNA using siRNA gandalfthegrey RNAi and SiRNA Forum 1 02-26-2008 04:32 PM
Rescue mRNA (reply to Sami) Jon D.Moulton Zebrafish Forum 0 05-08-2006 10:29 PM
New Saccharomyces Sequences 09/15/04 SGD Sequences Yeast Forum 0 09-15-2004 03:49 PM


All times are GMT. The time now is 08:07 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 0.18021 seconds with 16 queries