Real-Time PCR and Housekeeping gene - (Feb/26/2008 )

Hi all!
I've done a real-time PCR with 4 different housekeeping genes to selects which one are the best ones.
I've used cDNA from different tissue starting from the "same" amount of RNA (spectrophotometric analysis).
And now??? How can I compare this results??? I thought I could use the first housekeeping and use it as "housekeeping" to compare the other 3, than do the same thing with the second one and so on... but I'm not sure this is the correct way!
Does anyone have some clue how to do it?

Thanks

---

Hi Dott,

You have answered my question. Here's my opinion to yours.
A good housekeeping gene must not change its expression a lot in different conditions. So depends on what tissue you are using and what conditions you are studying, you can choose your HKG.
What are you studying?
1. a change in expression across different tissues with the same condition (say study same gene whose expression changes in the liver, heart, lung tissues) or
2. a change in expression in a type of tissue in different conditions (say treated and untreated liver, treated and untreated heart, treated and untreated lung tissue)?
I would say if 1, then you should quantify your rna or cDNA to the same concentration and see which HKG has the most stable expression (closest Cts) in all tissues.
If 2, then the HKG which has the closest Cts in all conditions (treated and untreated) should be chosen. Hope this helps.

Cheers,
Chris

---

Thanks Chris,

Now it's all clear!
I found out which is the best housekeeping to compare my different tissues (case 1), and now I'm running another plate on cells treated with different condition to find out which one is the best for this experiment too (case 2).

Bye
Berrino

---

If you want a more robust way of comparing reference genes than simply looking at Ct (it can be difficult to tell sometimes if you have a lot of samples), then I recommend using geNORM software, which is free and very easy to use. There is a website where you can download the software free, as well as instructions:

http://medgen.ugent.be/~jvdesomp/genorm/

A paper describing the principles is Vandesomple, et al., Genome Biology, 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.

---

Thanks grassgirl,
I already used GeNorm to compare my housekeeping genes, but now I'm wondering... cay I use it also to quantify my GOI?
I can't understand if it's possible and how to do it!! from the manual is not clear...
The real-time software (the one of my thermal cycler) can compare only GOI compared to 1 HK!
At the moment I have 3 HK and 1 GOI, how can I compare it (1 GOI) to 3 HK???

Thanks again

---

Hi Dott,

If you already found out which is the best HKG why fo you still need to compare 1 GOI with 3 HKG??

Chris

---

The software qBase allows you to compare GOI to several HK. This used to be a freeware, but is now a commercially available software (qBasePlus)

http://medgen.ugent.be/qbase/

Maybe it is still possible to find the free version somewhere

---

Hi Chris!
I'm new in Real-Time analysis... I'm just trying to do it in the "best" way, and also trying different ways.
I was told anaway is better to normalise against 2 or 3 different HKs instead of a single one.

Anaway I solved my problem, I found out it was only my fault ... my program is able to compare 1 GOI with 3 HKs... but I just gave wrong names to my samples (I used names like: Sample1-HK-A, Sample1-HK-B, Sample1-HK-C, Sample1-GOI... so the program didn't recognised them as the same sample and wasn't able to analyze them!!!)
It just a stupid mistake... but "you can always learn from your mistakes!!!"

Thanks to you also boxfish!!!

---

you're right to still use 3 HKG, any less than that is slightly dodgy...

---

The new qBasePlus software is the professional successor of the old qBase software, and is developed and supported by Biogazelle. There is an academic and commercial license for a reasonable fee, as well as a free license with limited functionality. There are a lot of improvements in qBasePlus compared to qBase, an overview is available here.

---