general QPCR problem

[bravebeaker]

Hi people,
First, I want to sincerely apologize if the problem have been covered else where, but I didnt find any while searching!
My problem is with QPCR experiment, I am using Roche Light Cycler 480.

I am testing a gene of interest against Actin (house keeping gene).
My control group which should and I know it doesnt contain much of my gene of interest is giving high readings in comparison to treatments with high induction of my gene of interest.

I repeated the experiments many times with almost the same result, control group is not near zero as it should be.

Any troubleshooting would be highly appreciated.

[greatmicrobiologist]

i feel like that the house keeping gene, Actin you are using is having some genomic sequences that is matching with your gene of interest. So my question is are you sure about the proper sequence of actin you are using?

[bravebeaker]

thanks for the reply
to the best of my knowledge the Actin primer is designed properly how would I double check the sequence?

[merv]

Hi people,
First, I want to sincerely apologize if the problem have been covered else where, but I didnt find any while searching!
My problem is with QPCR experiment, I am using Roche Light Cycler 480.

I am testing a gene of interest against Actin (house keeping gene).
My control group which should and I know it doesnt contain much of my gene of interest is giving high readings in comparison to treatments with high induction of my gene of interest.

I repeated the experiments many times with almost the same result, control group is not near zero as it should be.

Any troubleshooting would be highly appreciated.

I have no qPCR experience, so check with Roche and your supervisor as well. But maybe in these comments you will find something that will help:

So first of all you need to get your controls working as I know you dont want to waste time and reagents with your test gene.

Which actin are you using - alpha actin i recall is the frequently used one. Have your primers been designed by you (clearly not, so how can you trust them) and are they fresh and have you determined the ideal quantity to use in the reaction? Are these labelled primers, and how were they designed? it is important to design your primers so that they do not bind non-specifically (how long, what GC content, what predicted melting temperature. you should have considered all of this - also have you run the primers through the genbank database to see if there is any overlap with genes which your cell may also be making, which may or may not be homologs or spliced variants (have you looked at the genomic sequence and the cDNA) , and when you say your gene is not up without treatment how do you know, because if it is by antibody then you may have missed alternative spliced variants.

How are you making your message, as the other commentator wrote, you may be amplifying from genomic DNA (gDNA). This is one of the reasons for the actin control, so it is vital that you get a sample free of this if this is a problem. Also are you doing a one tube qRT-PCR or a clean-up between your RNA prep and your PCR. If you are going straight through without making a clean cDNA prep, then if things go wrong it is impossible to know at which stage you are having problems. Ideally, you should probably make a cDNA prep which you can freeze and go back to many times, using a tiny aliquot each time - a fresh prep cannot be re-used as it cannot be stored in the freezer- even in a -80oC freezer an RNA prep will degrade on ice and in the freeze/thaw cycles.

How are you counting your cells? How do you know they are healthy?
this is important as you have to be able to prove that you are using the same amount of mRNA from your cells - you should split the cell prep and make total DNA and RNA to be sure of the number of cells employed in your study.
(e.g. if your treating of your cells causes cell death or wholescale downregulation of mRNA then you have a problem as you are trying to do quantative PCR and this is meaningless without a useful control as you will know).
Also, why have you chosen actin? there are many so-called housekeeping genes, but although many people only compare to actin, some treatments change the amount of actin as well (for example, if you don't compare total RNA and/or DNA.)

good luck!

[bravebeaker]

merv merv merv!
Thanks alot for the comprehensive troubleshooting notes.
Yes you are right, I didnt design the primers however I tried the same experiment with a different gene of interest and I got the control result I was looking for.
I am using actin for plant cell genome as its the most favorable one in our lab.
I prepare cDNA after isolating RNA and I keep my cDNA frozen (-30C).
How did I expect my control result? From already nothern blotting reports, however I still believe that even in the case of no band in nothern blotting its not necessary for qPCR to be absolutely zero but at the sametime its impossible to be as the same as the induction treatment. Am I right?
For both genes of interest I followed the same techniques and procedures and I am concluding that I have a problem with the design of the first set of primers. Will go through your points again for a new set and will get back to you.

Again, thanks alot!

[JackBean]

How are you making your message, as the other commentator wrote, you may be amplifying from genomic DNA (gDNA). This is one of the reasons for the actin control, so it is vital that you get a sample free of this if this is a problem.

That's not true. The actin is used to compare different levels of mRNA.
Not to amplify from gDNA, one should design primers on exon border, so that you get no amplicon from gDNA.

[merv]

How are you making your message, as the other commentator wrote, you may be amplifying from genomic DNA (gDNA). This is one of the reasons for the actin control, so it is vital that you get a sample free of this if this is a problem.

That's not true. The actin is used to compare different levels of mRNA.
Not to amplify from gDNA, one should design primers on exon border, so that you get no amplicon from gDNA.

So sorry to have to correct you there JackBean - if gDNA is in the sample, then you are at risk of false positive from genomic , regardless of how you design the primers. You will find that exons are in the message as well...

[merv]

merv merv merv!
Thanks alot for the comprehensive troubleshooting notes.
Yes you are right, I didnt design the primers however I tried the same experiment with a different gene of interest and I got the control result I was looking for.
I am using actin for plant cell genome as its the most favorable one in our lab.

then the primers should be well tested. Also you should have some RNA from previous experiments with a known amount of RNA which gives you the standard result and you need to show that in your hands you are getting the same result as others. Only then will your experimental sample-result have any meaning whatsoever.

I prepare cDNA after isolating RNA and I keep my cDNA frozen (-30C).

thats fine for cDNA. Do you isolate total RNA or message alone, and do you perform any control primers to test for genomic actin?

How did I expect my control result? From already nothern blotting reports, however I still believe that even in the case of no band in nothern blotting its not necessary for qPCR to be absolutely zero but at the sametime its impossible to be as the same as the induction treatment. Am I right?

I dont really understand - in my opinion if there is no band in northern blotting, then you are probably using the wrong technique. does it work in northern blotting for your control? if it does then thats enough of a result, and you dont need to do the pcr. Also, you havent convinced me that the primers worked in a normal PCR, so why are you doing the qPCR?

For both genes of interest I followed the same techniques and procedures and I am concluding that I have a problem with the design of the first set of primers. Will go through your points again for a new set and will get back to you.

did you dilute your primers properly?

[bravebeaker]

thats fine for cDNA. Do you isolate total RNA or message alone, and do you perform any control primers to test for genomic actin?

I isolate total RNA and I dont use control primers to test for genomic actin but during my reverse trascriptase step I prepare -ve controls to check for contamination, and my -ve controls are fine.

I dont really understand - in my opinion if there is no band in northern blotting, then you are probably using the wrong technique. does it work in northern blotting for your control? if it does then thats enough of a result, and you dont need to do the pcr. Also, you havent convinced me that the primers worked in a normal PCR, so why are you doing the qPCR?

You are propably right about if I need to perform a qpcr or not but I want to double check and compare results as one is qualitative and the other one is quantitative. About my primers.. I use the same primers for PCR and QPCR and the primers are diluted to prepare 10pmol (10uM) conc.

I already designed a new set of primers I am going to test soon, will keep you posted with the results.

[JackBean]

I have no qPCR experience

How are you making your message, as the other commentator wrote, you may be amplifying from genomic DNA (gDNA). This is one of the reasons for the actin control, so it is vital that you get a sample free of this if this is a problem.

That's not true. The actin is used to compare different levels of mRNA.
Not to amplify from gDNA, one should design primers on exon border, so that you get no amplicon from gDNA.

So sorry to have to correct you there JackBean - if gDNA is in the sample, then you are at risk of false positive from genomic , regardless of how you design the primers. You will find that exons are in the message as well...

Of course the exons are in mRNA, that's what is mRNA composed of, isn't it? But if you designe forward primer in exon 1 and reverse primer in exon 2, you will get amplicon from mRNA (cDNA), but not from gDNA, because the intron is too long to be amplified during qPCR cycles.

Let's assume the actin is to check for gDNA contamination. How do you want to distinguish the contaminated sample from the one which isn't?