4 replies to this topic

[JackieG]

So, My approach to quantifying my transcript levels currently is this:

I use a standard curve (for every run) against which I let the software that came with our machine to quantify the amount of transcript levels in each of my samples.

I use the same method ("absolute" quantification against a standard curve) for my housekeeping gene


Ignoring the issues of amplification efficiency for each primer set ( my efficiencies are very close approx 1.98-2), is it correct to then simpy divide my target gene values by my housekeeping values, and call this a fold difference in transcript levels of gene X compared to housekeeping gene.


My confusion is coming from the fact that I haven't seen very many papers quantify their RNA prior to dividing by the housekeeping gene (instead use Ct values), though this makes the most intuitive sense to me.

Can some one help me with this concept?

[Trof]

This ABI User Bulletin explains relative quantification using a standard curve and says:

Quote

Divide the amount of c-myc by the amount of GAPDH to determine the normalized
amount of c-myc

(where c-myc is a target and GAPDH reference)

 ABI_Bulletin_2_Relative_Quantification.pdf   483.22K   99 downloads

[JackieG]

Trof, on May 7 2010, 09:07 AM, said:

This ABI User Bulletin explains relative quantification using a standard curve and says:

Quote

Divide the amount of c-myc by the amount of GAPDH to determine the normalized
amount of c-myc

(where c-myc is a target and GAPDH reference)

 ABI_Bulletin_2_Relative_Quantification.pdf   483.22K   99 downloads

Thanks alot I'll check it out~

[JackieG]

JackieG, on May 7 2010, 09:11 AM, said:

Trof, on May 7 2010, 09:07 AM, said:

This ABI User Bulletin explains relative quantification using a standard curve and says:

Quote

Divide the amount of c-myc by the amount of GAPDH to determine the normalized
amount of c-myc

(where c-myc is a target and GAPDH reference)

 ABI_Bulletin_2_Relative_Quantification.pdf   483.22K   99 downloads

Thanks alot I'll check it out~

OK, so I have checked out this method, but I am a bit confused about the calibrator step.


It says:

Step 1. Average the c-myc and GAPDH values from Table 1.
2. Divide the c-myc average by the GAPDH average.
3. Designate the calibrator.
4. Divide the averaged sample value by the averaged calibrator value.


What is the calibrator?

[JackieG]

JackieG, on May 8 2010, 06:34 PM, said:

JackieG, on May 7 2010, 09:11 AM, said:

Trof, on May 7 2010, 09:07 AM, said:

This ABI User Bulletin explains relative quantification using a standard curve and says:

Quote

Divide the amount of c-myc by the amount of GAPDH to determine the normalized
amount of c-myc

(where c-myc is a target and GAPDH reference)

 ABI_Bulletin_2_Relative_Quantification.pdf   483.22K   99 downloads

Thanks alot I'll check it out~

OK, so I have checked out this method, but I am a bit confused about the calibrator step.


It says:

Step 1. Average the c-myc and GAPDH values from Table 1.
2. Divide the c-myc average by the GAPDH average.
3. Designate the calibrator.
4. Divide the averaged sample value by the averaged calibrator value.


What is the calibrator?

Nevermind, got it! Its totally arbitrary