v***a
发帖数: 1242 | 1 做overexpression，把细胞terminate后提取RNA做RT-PCR，S16做内参。PCR结果出来一
看，发现目的基因都有P出来，overexpression的比vehicle ctrl目的基因的band强烈
一点，但S16的band淡到几乎看不出。vehicle control的S16就很亮。
我取了好几个time point的，每个time point都是有overexpression的S16很淡。
请教一下牛人这是为什么啊？是不是也没法定量比较是否overexpression成功了？ |
s******y
发帖数: 28562 | 2 Did you transfect your cell with a plasmid?
In such a case, you need to treat the RNA sample carefully with DNaseI to
remove all the plasmid DNAs. Otherwise you are just doing PCR using the
plasmid as template
【在 v***a 的大作中提到】
: 做overexpression，把细胞terminate后提取RNA做RT-PCR，S16做内参。PCR结果出来一
: 看，发现目的基因都有P出来，overexpression的比vehicle ctrl目的基因的band强烈
: 一点，但S16的band淡到几乎看不出。vehicle control的S16就很亮。
: 我取了好几个time point的，每个time point都是有overexpression的S16很淡。
: 请教一下牛人这是为什么啊？是不是也没法定量比较是否overexpression成功了？
|
v***a
发帖数: 1242 | 3 谢谢mm回答。
是的，我没有用。。。打算重做了。
但我想不通这个跟我的S16有什么关系呢
【在 s******y 的大作中提到】
: Did you transfect your cell with a plasmid?
: In such a case, you need to treat the RNA sample carefully with DNaseI to
: remove all the plasmid DNAs. Otherwise you are just doing PCR using the
: plasmid as template
|
v***a
发帖数: 1242 | 4 请问一下，MM你的DNASE I是哪个公司买的？为什么一定要用这个处理呢？提取RNA以后
还是会残留很多DNA吗？
【在 s******y 的大作中提到】
: Did you transfect your cell with a plasmid?
: In such a case, you need to treat the RNA sample carefully with DNaseI to
: remove all the plasmid DNAs. Otherwise you are just doing PCR using the
: plasmid as template
|
s******y
发帖数: 28562 | 5 Depending on your purification methods, but I recommend the
RNase-free DNase kit from Qiagne.
It is CRITICAL to remove the Plasmid DNA, otherwise you are just doing PCR
on your plasmid, not the cDNA. Most RNA purification kit are designed to
remove genomic DNA but they are NOT designed to remove plasmid DNA,
therefore you have to do it yourself.
The reason you don't see the S16 can be due to:
1. you don't add enough cDNA template (plasmid DNA has strong asorption and
will mislead you to think yo
【在 v***a 的大作中提到】
: 请问一下，MM你的DNASE I是哪个公司买的？为什么一定要用这个处理呢？提取RNA以后
: 还是会残留很多DNA吗？
|
v***a
发帖数: 1242 | 6 Thank you very much!!!
I totally understand now.
and
【在 s******y 的大作中提到】
: Depending on your purification methods, but I recommend the
: RNase-free DNase kit from Qiagne.
: It is CRITICAL to remove the Plasmid DNA, otherwise you are just doing PCR
: on your plasmid, not the cDNA. Most RNA purification kit are designed to
: remove genomic DNA but they are NOT designed to remove plasmid DNA,
: therefore you have to do it yourself.
: The reason you don't see the S16 can be due to:
: 1. you don't add enough cDNA template (plasmid DNA has strong asorption and
: will mislead you to think yo
|
r**o
发帖数: 212 | 7 Good points.
and
【在 s******y 的大作中提到】
: Depending on your purification methods, but I recommend the
: RNase-free DNase kit from Qiagne.
: It is CRITICAL to remove the Plasmid DNA, otherwise you are just doing PCR
: on your plasmid, not the cDNA. Most RNA purification kit are designed to
: remove genomic DNA but they are NOT designed to remove plasmid DNA,
: therefore you have to do it yourself.
: The reason you don't see the S16 can be due to:
: 1. you don't add enough cDNA template (plasmid DNA has strong asorption and
: will mislead you to think yo
|
