Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

Analysis HELP!!!! - (Apr/29/2010 )

Hi all,

I am a newbie in this forum as you can see from the number of my posts. So here it goes. Let me start by saying that I work with a very small protist and that not very much is known about this organism's metabolic pathways at all. I am exploring expression of a few genes under 4 different conditions. I am using 3 housekeeping genes. I have optimized conditions for all my target and HK genes, have brought DNA contamination to an absolute minimum, normalized everything and basically have tried to eliminate as much error as possible. I have also tested the suitability of all 3 HK genes. So I am ready to go!

This is a one time experiment, it is not something that I have to do on routinely. So my method of choice was the relative quantification using standard curves for the HK genes and the target genes every time. The cycler I use (Realplex) does not have the software to calculate this and so I have to do this myself. So basically what I do is

target gene copy #target/copy #control=A
HK gene copy #target/copy #control=B

Then simply divide A/B. This gives me the fold change of the target relative to the HK.

My question is if this calculation can give a down-regulation. I mean I can tell if the change is 8 fold for instance that my gene of interest has an 8fold increase relative to the HK. But what if the gene is down-regulated? Would I then get values less than 1???? Any help would be greatly appreciated


Secondly and perhaps most importantly, I am faced with the problem of choosing an appropriate control. The reason is that we simply do not know which condition is control. We know for sure that certain genes are expressed in condition 1 (one extreme) but not in condition 4 (other extreme). Can I be switching control between conditions? My gut screams no to this (introducing bias, big time). Or alternatively can I do calculations using two different controls? Gut screams no again. Am I even making sense at all?

Any, any any help, input, comment would very much appreciated. Thanks

-ciliate-

ciliate on Apr 30 2010, 11:22 AM said:

Hi all,

I am a newbie in this forum as you can see from the number of my posts. So here it goes. Let me start by saying that I work with a very small protist and that not very much is known about this organism's metabolic pathways at all. I am exploring expression of a few genes under 4 different conditions. I am using 3 housekeeping genes. I have optimized conditions for all my target and HK genes, have brought DNA contamination to an absolute minimum, normalized everything and basically have tried to eliminate as much error as possible. I have also tested the suitability of all 3 HK genes. So I am ready to go!

This is a one time experiment, it is not something that I have to do on routinely. So my method of choice was the relative quantification using standard curves for the HK genes and the target genes every time. The cycler I use (Realplex) does not have the software to calculate this and so I have to do this myself. So basically what I do is

target gene copy #target/copy #control=A
HK gene copy #target/copy #control=B

Then simply divide A/B. This gives me the fold change of the target relative to the HK.

My question is if this calculation can give a down-regulation. I mean I can tell if the change is 8 fold for instance that my gene of interest has an 8fold increase relative to the HK. But what if the gene is down-regulated? Would I then get values less than 1???? Any help would be greatly appreciated


Secondly and perhaps most importantly, I am faced with the problem of choosing an appropriate control. The reason is that we simply do not know which condition is control. We know for sure that certain genes are expressed in condition 1 (one extreme) but not in condition 4 (other extreme). Can I be switching control between conditions? My gut screams no to this (introducing bias, big time). Or alternatively can I do calculations using two different controls? Gut screams no again. Am I even making sense at all?

Any, any any help, input, comment would very much appreciated. Thanks




im eppendolf realplex user too... calculate yourself? there is plenty of softwares that can help u with calculation (eg REST, GenEx...), so actually no need calculate urself de..
anyway, i do calculations myself too...
1. Normalized to housekeeping = (mean of Ct house keeping - Ct samples)
2. Relative mRNA expression to control = (normalized Ct samples - mean of normalized Ct control)
3. Relative mRNA expression to Control (means of 2 biological replicates)

then u can use the end result for plot the graph... (is it still confusing?) ...

hopefully it helps a bit...

-paramitay-