4 replies to this topic

[novagen]

Hi all.

I want to study expression levels of a gene in different parts of an organism having (different expression levels in ) but unfortunately My lab doesnt have Real time RT-PCR. So, Is there any other alternative for studying expression levels using cDNA of that gene.

is anyone working with semi-quantitative RT-PCR.
ThanX in advance

[cellcounter]

novagen, on Jul 22 2009, 09:29 AM, said:

Hi all.

I want to study expression levels of a gene in different parts of an organism having (different expression levels in ) but unfortunately My lab doesnt have Real time RT-PCR. So, Is there any other alternative for studying expression levels using cDNA of that gene.

is anyone working with semi-quantitative RT-PCR.
ThanX in advance

(1) Semi-quantitaive RT-PCR was the standard when there was no real-time PCR. So, you can fall back on it. Do different numbers of PCR cycles or serial dilutions of first strands. Make sure to design intron-spanning primers.

(2) Westerns (ELISA if applicable).

(3) Microarrays!

[novagen]

Hi Cellcounter,

Thanx a lot for ur reply
The only option left for me is semi-quantitative RT-PCR.
Can you please describe it in detail.

THanX

[cellcounter]

novagen, on Jul 22 2009, 07:43 PM, said:

Hi Cellcounter,

Thanx a lot for ur reply
The only option left for me is semi-quantitative RT-PCR.
Can you please describe it in detail.

THanX

Basically, you do first strand synthesis (RT) followed by routine PCR. But to quantitate the amount of transcript, you either compare the PCR product amount with an endogenous housekeeping gene (one of many that have been found to be similar in expression level across many tissues or cell lines), or better still, add an exogenous standard template (synthetic - you create it) that allows you to amplify a different size of product using the same primers as your gene specific PCR.

Then you do your PCR reaction using several different numbers of amplification cycles (20-24-28-32 etc), or several different dilutions, and make sure that you are getting a linear amplification (just like real-time, but crude and manual) and are not reaching a saturation level. And then you compare the product amounts from RNA isolated from various tissues.

There is a lot to be said about the overall method,  and there are several ways to do this -ranging from very simple methods to quite complex and elaborate setups. What I wrote here is brief and does not do justice to the methods. I suggest some reference reading on this subject. To start you, here are some protocols that will give you an idea of the wide range that this term encompasses.

[novagen]

Hi cellcounter
Thank you very very much........ for your suggestion
Thats a great website
I will go through and will definately come back to U