New to Real Time, please help - (Aug/16/2007 )

Hello everyone,

Iam new to real time..I have run my samples using sybr green, however for my house keeping genes iam getting great variability (not between triplicates) but between samples?my HKG are bactin and gapdh.

Is there a way to reduce this?

And also, what kind of difference is considered significant between house keeping genes? a ct difference of 2? or more?less?

Thanks

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Hi,

Based on my experience, it's usual to get a varied ct for both HKG (bactin & gapdh).that's why some of my friends they use ribosomal gen which they said, much more stable. In my work, I used bactin and I found out that you can reduce the variability by:

1) strictly look into the melt curve output. the same amount of template should result in a same melt curve output.then, if you still get a different ct (should not exceed 0.5 within samples) between samples (more than 2.0), that's mean it has something to do with your own experiment design.Anyway, when it comes to real calculation of fold changes, it is important to use target value and control value from same samples to reduce error.
2) difference of 2 indicate that both amplicons are 1 cycle apart.

sorry if it's not helping.

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Use primers for 18S rRNA. It's a polI transcript, and ribosomal rna's make up about 80% of total RNA from most cells. Your CT values may be VERY LOW for this gene however, so a 1:1000 dilution of your template might be required (depending on how rare your target gene is).

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Thank you both your help, its actually good to hear people having the same problems with real time samples


"strictly look into the melt curve output. the same amount of template should result in a same melt curve output.then, if you still get a different ct (should not exceed 0.5 within samples) between samples (more than 2.0), that's mean it has something to do with your own experiment design.Anyway, when it comes to real calculation of fold changes, it is important to use target value and control value from same samples to reduce error"

p38....that is exactly what Iam seeing..low varability within triplicates and high varability between samples...ct range =22 to 33 (all in different conditions/times), with identical melting curves.

If this is the result of the experiment itself, can these results still be interpreted? or are they simply artifacts? is what iam seeing real..

have you used these kind of values with variable HKG?

Thank you so much

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