reverse-transcriptase PCR (internal control?) - (Aug/12/2008 )
Hi, I am trying to get answer whether RT-PCR need the housekeeping genes as perform in Real Time PCR? pls help....
If yes, what is the purpose? Thanks in advance..
If yes, what is the purpose? Thanks in advance..
I think if you finally want to quantify your mRNAs, its necessary... At least in my case, I always use actin for normalization.
The controls are essential for loading quantity.
If you have equal distrbution of housekeeping gene(s) across your samples then you know that the total signal is unchanged. If for instance the controls don't work and you have 2x the housekeeping gene in one sample and not in the others, then you would consider that this sample has twice the signal and then the message for your favorite gene (YFG) would presumably be also double. Then you can normalize, if you're lazy, or alter the conditions so that you guarantee that you have equal saturation of message in all samples.
It's just like the loading controls for a western blot. If you have twice as much total protein then your experimental signal will be abnormal.
With RTPCR if you have twice the total mRNA your experimental mRNA will be abnormal and immiscible as scientific data.
If you have equal distrbution of housekeeping gene(s) across your samples then you know that the total signal is unchanged. If for instance the controls don't work and you have 2x the housekeeping gene in one sample and not in the others, then you would consider that this sample has twice the signal and then the message for your favorite gene (YFG) would presumably be also double. Then you can normalize, if you're lazy, or alter the conditions so that you guarantee that you have equal saturation of message in all samples.
It's just like the loading controls for a western blot. If you have twice as much total protein then your experimental signal will be abnormal.
With RTPCR if you have twice the total mRNA your experimental mRNA will be abnormal and immiscible as scientific data.
Hi everybody. I´m new in this forum!
I've a similar problem, I can´t amplify b-actin in my controls samples. I extract total RNA with Trizol (invitrogen) from macrophages, and then obtain cDNA. Then the first step is normailze the amount of message, I did it in my treated samples (T1, T2, T3) but i got problems with control samples. I´m attaching a gel pic to show that.
I wait your commets, Thank you!
Dr UDPG,
There are a number of possibilities:
1. Try to calibrate the reaction better (Tm or Mg2+)
2. Look in a primer database for a new b-actin primer
3. Use a different internal control (GAPDH, beta4 integrin or any other)
Dr UDPG,
can you amplify any other gene from your control sample? It looks from your gel like it isnt a problem of optimising PCR, as it works really nice for T1, T2, T3, but it might be a problem with your control RNA.
I'll check for that first, as you just have to run a PCR before redesigning primers, altering Temps, or playing with MgCl2 conc.
good luck!
can you amplify any other gene from your control sample? It looks from your gel like it isnt a problem of optimising PCR, as it works really nice for T1, T2, T3, but it might be a problem with your control RNA.
I'll check for that first, as you just have to run a PCR before redesigning primers, altering Temps, or playing with MgCl2 conc.
good luck!
Thank you very much guys for your comments. Sometimes I can amplify IL1 or IL6 genes in control samples. Does Trizol make a differential extractions of total RNA? T1, T2 and T3 are cDNA copy of macrophages treated with snake venoms. I´ve allready seen in other posts the same problems with controls, that's why i´m asking ...
Thank you!
Dr. UDPG