qRT-PCR

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Zelda
Zelda's picture
qRT-PCR

 Hello all. I am back again with a new question.
I started my RT-PCR but I wasconfused with somthing..
I do have 2 house keeping gene, two tissue and4 genes (8 primers)
I want to do 4 dilution and undiluted sample
which sample should assigned as standered and unkown to have a standered curve
and what is the intial concentration for each  sample diluted and undiluted as I was asked by the program
I need help please as soon as possible
do you know any helpful website as well

thank and all

Ivan Delgado
Ivan Delgado's picture
Hi Zelda,

Hi Zelda,

You should run a standard curve for each assay you will be testing, so if you have 4 genes then you need 4 standard curves to quantify each gene. 

The initial concentration of each sample depends on how sensitive your assay is, which you will determine once you run your standard curves. If your standard curves are able to detect 10 nanograms, then that is your initial dilution. 

Good luck!

Zelda
Zelda's picture
 Hi thank you for your reply.

 Hi thank you for your reply.

Can I consider my house keeping gene as a standard
and do I need to use dilution with my standard gene as well 

Zelda
Zelda's picture
 When I set the samples in

 When I set the samples in the machine it say standard sample should have concentration above zero
and I get a box with the following equation 1.E+06 
what is this equation and how i use it to proced with next step

Ivan Delgado
Ivan Delgado's picture
It depends on what standard

It depends on what standard you are using. If you are quantifying based on a standard curve, then you need to use every gene you are studying as a standard, and make a dilution series for every gene. 

1.E+06 mean 1 followed by 6 zeros, so 1 million. A standard sample does not need to be any amount. Likely you are seeing this in the machine as a default number. The amounts you use for your standard curve are amounts you need to determine based on how sensitive your assay is. Just run a number of dilutions and see which ones work best. 

Zelda
Zelda's picture
 Thank you so much. 

 Thank you so much. 
Should I do the dilution curve with each tissue I am using or one is enough.
Thanks again :)

Ivan Delgado
Ivan Delgado's picture
It depends. Most people would

It depends. Most people would say that once you've run a standard curve with one tissue that is enough since the assay has already been shown to work properly with one tissue. Having said that others would say that the assay behaves differently when the RNA comes from different tissues so you should run standard curves for every tissue. In short, I would suggest just running the standard curve with one tissue and assume that this is good enough for all tissues. 

msdv
msdv's picture
hi ,

hi ,
can any one help me out ? i am doing real time with heart tissue. in order to get good results in normalising the std . curve of HKG what concentration of cDNA should i use. I have 7 HKG and initially i used 50ngs of cDNA and i obtained good results for 4 HKG whereas for the other 3 i dont get ! should i use increased concentration of cDNA or what should i do ?

Ivan Delgado
Ivan Delgado's picture
Increasing the amount of cDNA

Increasing the amount of cDNA would be a good place to start. If you cannot get good results for the other 3 HKGs after  you increase the amount of cDNA you are using, you may want to consider using other HKGs. 

Good luck!

msdv
msdv's picture
thanks a lot

thanks a lot