a*****s
发帖数: 131 | 1 Hello everyone, please help: how to detect low abundance gene expression?
Now I use 1 ug of RNA per 20 ul cDNA reaction, then I use 1 ul of the cDNA.
For beta-Actin, the qPCR picks up the signal at around 15 cycles in linear
range. But my target gene (I used 2.5 ul per reaction) can only be picked up
at around 35 cycles (1 or 2 out of triplicated reactions), where it is
already out of the linear range!! How to enhance the signal?? Please help!
Thank you very much!! So frustrated... |
F*K
发帖数: 608 | 2 do not force it, the gene is simply not expressed.
.
up
【在 a*****s 的大作中提到】
: Hello everyone, please help: how to detect low abundance gene expression?
: Now I use 1 ug of RNA per 20 ul cDNA reaction, then I use 1 ul of the cDNA.
: For beta-Actin, the qPCR picks up the signal at around 15 cycles in linear
: range. But my target gene (I used 2.5 ul per reaction) can only be picked up
: at around 35 cycles (1 or 2 out of triplicated reactions), where it is
: already out of the linear range!! How to enhance the signal?? Please help!
: Thank you very much!! So frustrated...
|
m**********2
发帖数: 6568 | 3 you need to have no-RT controls. If no-RT control has a much higher Ct (say
45) then you are fine. If no-RT is also 35, then forget it. Just say your
gene is not detectable under the condition.
.
up
【在 a*****s 的大作中提到】
: Hello everyone, please help: how to detect low abundance gene expression?
: Now I use 1 ug of RNA per 20 ul cDNA reaction, then I use 1 ul of the cDNA.
: For beta-Actin, the qPCR picks up the signal at around 15 cycles in linear
: range. But my target gene (I used 2.5 ul per reaction) can only be picked up
: at around 35 cycles (1 or 2 out of triplicated reactions), where it is
: already out of the linear range!! How to enhance the signal?? Please help!
: Thank you very much!! So frustrated...
|
a*****s
发帖数: 131 | 4 No-RT controls have no amplifications until 45 cycles... so I believe the
gene is expressed...
Thanks for more suggestions!
say
【在 m**********2 的大作中提到】
: you need to have no-RT controls. If no-RT control has a much higher Ct (say
: 45) then you are fine. If no-RT is also 35, then forget it. Just say your
: gene is not detectable under the condition.
:
: .
: up
|
r******8
发帖数: 110 | 5 What's No-RT controls ?Is the RNA without reverse-transcription?
【在 a*****s 的大作中提到】
: No-RT controls have no amplifications until 45 cycles... so I believe the
: gene is expressed...
: Thanks for more suggestions!
:
: say
|
l***y
发帖数: 638 | 6 pre-amp your sample for 10-15 cycles, that will bring down the Ct of your
gene to 20ish, but you might need to find a different housekeeping gene to
normalize to because Ct of actin will be below 10.
.
up
【在 a*****s 的大作中提到】
: Hello everyone, please help: how to detect low abundance gene expression?
: Now I use 1 ug of RNA per 20 ul cDNA reaction, then I use 1 ul of the cDNA.
: For beta-Actin, the qPCR picks up the signal at around 15 cycles in linear
: range. But my target gene (I used 2.5 ul per reaction) can only be picked up
: at around 35 cycles (1 or 2 out of triplicated reactions), where it is
: already out of the linear range!! How to enhance the signal?? Please help!
: Thank you very much!! So frustrated...
|
m**********2
发帖数: 6568 | 7 in this case I'd suggest you do a few 2x dilutions of the cDNA and see if
you get a linear curve with the Ct. If that is good then pretty convincing
to me.
【在 a*****s 的大作中提到】
: No-RT controls have no amplifications until 45 cycles... so I believe the
: gene is expressed...
: Thanks for more suggestions!
:
: say
|
