h*********g
发帖数: 18 | 1 我作的qPCR，一个基因的cycles在control条件是36 or 37 cycles，在刺激下的cycles
是 30 cycles。请问这是否表明此基因表达升高很多倍？好像35个cycles以上就不准了
？多谢！ |
t******k
发帖数: 5617 | 2 看melting curve是不是expectedproduct，ddCt的话，37到30是2^7倍，dramatic
change. |
A******y
发帖数: 2041 | 3 Not only that, is your primers still linear at cycle 30-36? Did you check
the linearity of the primers? I know a lot of people don't do qPCR
correctly. |
h*********g
发帖数: 18 | 4 I am using Taqman primer. Do you think there are some problems? Thanks.
cycles
【在 h*********g 的大作中提到】
: 我作的qPCR，一个基因的cycles在control条件是36 or 37 cycles，在刺激下的cycles
: 是 30 cycles。请问这是否表明此基因表达升高很多倍？好像35个cycles以上就不准了
: ？多谢！
|
A******y
发帖数: 2041 | 5 I don't care what primers that you are using. It never hurt to check the
linearity of your primers!
【在 h*********g 的大作中提到】
: I am using Taqman primer. Do you think there are some problems? Thanks.
:
: cycles
|
h*********g
发帖数: 18 | 6 Could you tell me how to check primer linearity? I did not find it on google.
Thanks!
【在 A******y 的大作中提到】
: I don't care what primers that you are using. It never hurt to check the
: linearity of your primers!
|
A******y
发帖数: 2041 | 7 So how much RNA or cDNA sample do you add to your qPCR? What you do is take
a sample (usually control), do a x2 serial dilution and see if your cycle
is close to a single cycle per dilution and if it is linear. It is call PCR
amplification efficiency. It doesn't have to 100% but I think you should
have >90% and linear. |
h*********g
发帖数: 18 | 8 I am using 10 ng cDNA in 10 ul. Thanks. I will try.
take
PCR
【在 A******y 的大作中提到】
: So how much RNA or cDNA sample do you add to your qPCR? What you do is take
: a sample (usually control), do a x2 serial dilution and see if your cycle
: is close to a single cycle per dilution and if it is linear. It is call PCR
: amplification efficiency. It doesn't have to 100% but I think you should
: have >90% and linear.
|
h*********g
发帖数: 18 | 9 Here is my cycle numbers corresponding to 2X dilution. How do you think?
Thanks.
28.8, 30.13, 31, 32.74, 33.86
I do not have positive control and can only use my sample.
take
PCR
【在 A******y 的大作中提到】
: So how much RNA or cDNA sample do you add to your qPCR? What you do is take
: a sample (usually control), do a x2 serial dilution and see if your cycle
: is close to a single cycle per dilution and if it is linear. It is call PCR
: amplification efficiency. It doesn't have to 100% but I think you should
: have >90% and linear.
|
A******y
发帖数: 2041 | 10 How about you tell me. If you draw a standard curve, is it linear? from 31
to 32.74, you have about 2 cycle difference and you only diluted by x2.
Unless you skip a point. You should also do more points.
【在 h*********g 的大作中提到】
: Here is my cycle numbers corresponding to 2X dilution. How do you think?
: Thanks.
: 28.8, 30.13, 31, 32.74, 33.86
: I do not have positive control and can only use my sample.
:
: take
: PCR
|
f*******e
发帖数: 628 | 11 通常 qPCR 会 report PCR efficiency. taqman 通常 efficiency 应该接近 100%. 如
果持续低于 95%， 说明 primer/probe design 有问题。
Ct大于30之后基本上就不太reliable了。做了no template control么? 离你的sample
的 Ct远么？ |
F******p
发帖数: 2099 | 12 >32 is not reliable.
I suspect you were using 2 primers qPCR. Try 2 primers and probe based qPCR
, that is way more reliable.
cycles
【在 h*********g 的大作中提到】
: 我作的qPCR，一个基因的cycles在control条件是36 or 37 cycles，在刺激下的cycles
: 是 30 cycles。请问这是否表明此基因表达升高很多倍？好像35个cycles以上就不准了
: ？多谢！
|
A******y
发帖数: 2041 | 13 I am just sad that the PI don't train people properly, you should always
validate your primer regardless what people told you. If you have controls,
high cycle range is not a problem. |
