t*x
发帖数: 1065 | 1 半年前用相同的primer,cDNA synthesis kit做的，当时cDNA只用到了0.1ug/reaction.
半年之后其他东西几乎是相同的，只不过RNA是新提的（但用的相同的kit),还有SYBR
Green Master Mix是新买的，但发现，cDNA已经用到了很高的浓度(2ug/reaction)，但
Ct值还是比以前的Ct值晚出现好几个cycle,请问会是什么原因呢？
RNA quality已经跑胶检测过了，是完整的。
会是Master Mix的问题么？是新买的啊，一直保存在-20C.
多谢大家指教！ |
U********S
发帖数: 1896 | 2 housekeeping gene 的Ct相差达吗？ |
t*x
发帖数: 1065 | 3 对的，18SrRNA的 Ct也相差很大。。。今天又重新做了一遍，还是这样的结果
碰到过相似情况么？？
谢谢哦～～
【在 U********S 的大作中提到】
: housekeeping gene 的Ct相差达吗？
|
U********S
发帖数: 1896 | 4 RNA定量准的话换个RT kit先。
【在 t*x 的大作中提到】
: 对的，18SrRNA的 Ct也相差很大。。。今天又重新做了一遍，还是这样的结果
: 碰到过相似情况么？？
: 谢谢哦～～
|
s******y
发帖数: 28562 | 5 well, if the relative ct (your target gene over the house keeping gene)
remain the same, then there is nothing to worry about.
If you really really want to make sure, there are things you need
to check:
1. did they change the fluorescent lamp/detector in the realPCR machine?
2. Did you use the same DNA dye in the PCR reaction? (if the dye is loaded
in the premix kit, then it is not consistent between batches and cannot be
used to do direct comparison)
reaction.
【在 t*x 的大作中提到】
: 半年前用相同的primer,cDNA synthesis kit做的，当时cDNA只用到了0.1ug/reaction.
: 半年之后其他东西几乎是相同的，只不过RNA是新提的（但用的相同的kit),还有SYBR
: Green Master Mix是新买的，但发现，cDNA已经用到了很高的浓度(2ug/reaction)，但
: Ct值还是比以前的Ct值晚出现好几个cycle,请问会是什么原因呢？
: RNA quality已经跑胶检测过了，是完整的。
: 会是Master Mix的问题么？是新买的啊，一直保存在-20C.
: 多谢大家指教！
|
t*x
发帖数: 1065 | 6 Thanks a lot!!! Really appreciate!
You are totally right....The relative Ct seems remain same and the dye
actually is added together with the premix. The reason why I worry this so
much is that the Ct value of my target gene is so late (>35) even though I'
ve already increase the concentration of cDNA so much. |
t*x
发帖数: 1065 | 7 Thanks. I changed but results remain similar.
Is it okay that I load 2-4ug cDNA/reaction? I am afraid it is too much and
is so abnormal. |
b****x
发帖数: 110 | 8 I think it is not necessary to load 2-4ug cDNA. It is too much even if you
get a relatively normal Ct value.
Checking the machine settings for all parameters is helpful. |
t*x
发帖数: 1065 | 9 Thanks for your response.
Yeah, I also concern about that. But if I load less cDNA, there is no
amplification curve before 40 cycles at all.
The machine settings did not change at all, but I'll double check. Thanks!
【在 b****x 的大作中提到】
: I think it is not necessary to load 2-4ug cDNA. It is too much even if you
: get a relatively normal Ct value.
: Checking the machine settings for all parameters is helpful.
|
