v***a
发帖数: 1242 | 1 第一次做siRNA，想请教大家，用Trizol提取了RNA后，在进入RT-PCR之前有什么
注意事项吗？比如说像overexpression后要用Dnase I处理（感谢上次艳阳天mm的帮助
）。谢谢啊！ |
v***a
发帖数: 1242 | 2 在网上查了一下，貌似也要做Dnase I treatment，但想不通这是为什么呢
【在 v***a 的大作中提到】
: 第一次做siRNA，想请教大家，用Trizol提取了RNA后，在进入RT-PCR之前有什么
: 注意事项吗？比如说像overexpression后要用Dnase I处理（感谢上次艳阳天mm的帮助
: ）。谢谢啊！
|
a***e
发帖数: 1010 | 3 for any RT-PCR, you need a DNaseI pre-treatment. |
v***a
发帖数: 1242 | 4 谢谢。以前只做从tissue中提取RNA，都没做过DNase I pre-treatment。出来的RT-PCR
的data倒是跟IB的吻合的。
不知道为什么in vitro了就不可以了呢？
还有，上次艳阳天mm说overexpression后做DNase I treatment是为了去除plasmid DNA
，想不通这个siRNA后是为了去除什么？谢谢。。。
【在 a***e 的大作中提到】
: for any RT-PCR, you need a DNaseI pre-treatment.
|
h****g
发帖数: 439 | 5 因为用Trizol或者说任何方法提RNA，都不能保证没有genomic DNA的contamination。所以要在RT前做一下 DNase I treatment
另外一个方法是，设计的引物要跨Intron，这样也可以避免genomic DNA 的contamination 的干扰.
PCR
DNA
【在 v***a 的大作中提到】
: 谢谢。以前只做从tissue中提取RNA，都没做过DNase I pre-treatment。出来的RT-PCR
: 的data倒是跟IB的吻合的。
: 不知道为什么in vitro了就不可以了呢？
: 还有，上次艳阳天mm说overexpression后做DNase I treatment是为了去除plasmid DNA
: ，想不通这个siRNA后是为了去除什么？谢谢。。。
|
v***a
发帖数: 1242 | 6 谢谢
那这么说我之前从tissue中提的没做DNase I treatment的result不太可靠喽？
设计引物我一般是在CDS区选，这样应该没问题吧
。所以要在RT前做一下 DNase I treatment
contamination 的干扰.
【在 h****g 的大作中提到】
: 因为用Trizol或者说任何方法提RNA，都不能保证没有genomic DNA的contamination。所以要在RT前做一下 DNase I treatment
: 另外一个方法是，设计的引物要跨Intron，这样也可以避免genomic DNA 的contamination 的干扰.
:
: PCR
: DNA
|
n****n
发帖数: 165 | 7 Don't worry about DNA. Because your control and KD sample have the same
treatment and you just compare them, it doesn't matter whether you do DNAase
treatment or mot. |
a***e
发帖数: 1010 | 8 No, it does matter.
The contamination of DNA in the RNA sample will mask the fold change
caused by RNAi.
For example, your RNA is 10 before treatment, but 1 after treatment. Thus
the fold change is 10.
However, if you have 5 part of DNA in your sample, the fold change 15/6 =
2.5 fold.
DNAase
【在 n****n 的大作中提到】
: Don't worry about DNA. Because your control and KD sample have the same
: treatment and you just compare them, it doesn't matter whether you do DNAase
: treatment or mot.
|
f*********r
发帖数: 1233 | 9 不用这么小心翼翼。
如果WB显示KD了50-80%，RNA应该没啥问题。跑一个RT-PCR看看就是了。如果KD水平没
有达到90%以上，再去考虑加DNase。
如果WB显示KD水平小于50%，甚至不足1/3，就不用琢磨RNA了。 |
v***a
发帖数: 1242 | 10 thank you
pretty clear
【在 a***e 的大作中提到】
: No, it does matter.
: The contamination of DNA in the RNA sample will mask the fold change
: caused by RNAi.
: For example, your RNA is 10 before treatment, but 1 after treatment. Thus
: the fold change is 10.
: However, if you have 5 part of DNA in your sample, the fold change 15/6 =
: 2.5 fold.
:
: DNAase
|
|
|
v***a
发帖数: 1242 | 11 谢谢！
【在 f*********r 的大作中提到】
: 不用这么小心翼翼。
: 如果WB显示KD了50-80%，RNA应该没啥问题。跑一个RT-PCR看看就是了。如果KD水平没
: 有达到90%以上，再去考虑加DNase。
: 如果WB显示KD水平小于50%，甚至不足1/3，就不用琢磨RNA了。
|
c**n
发帖数: 73 | 12 The easier way is to design a pair of primers at exon junctions so that the
PCR reaction won't pick up anything from genomic DNA.
You can certainly do DNase I treatment but often you lose a lot of RNA
during this step.
【在 v***a 的大作中提到】
: 谢谢
: 那这么说我之前从tissue中提的没做DNase I treatment的result不太可靠喽？
: 设计引物我一般是在CDS区选，这样应该没问题吧
:
: 。所以要在RT前做一下 DNase I treatment
: contamination 的干扰.
|
y****i
发帖数: 2194 | 13 Dude, genomic DNA has only two copies/per cell.
Thus
=
【在 a***e 的大作中提到】
: No, it does matter.
: The contamination of DNA in the RNA sample will mask the fold change
: caused by RNAi.
: For example, your RNA is 10 before treatment, but 1 after treatment. Thus
: the fold change is 10.
: However, if you have 5 part of DNA in your sample, the fold change 15/6 =
: 2.5 fold.
:
: DNAase
|
n****n
发帖数: 165 | 14 Maybe you are right. But we do this so many times without DNAase treatment
and always get not bad results for Real-time PCR. So i think treatment with
DNAase or not is not big problem.
【在 a***e 的大作中提到】
: No, it does matter.
: The contamination of DNA in the RNA sample will mask the fold change
: caused by RNAi.
: For example, your RNA is 10 before treatment, but 1 after treatment. Thus
: the fold change is 10.
: However, if you have 5 part of DNA in your sample, the fold change 15/6 =
: 2.5 fold.
:
: DNAase
|
h********n
发帖数: 4079 | 15 I run no-RT control.
I don't think DNase is REQUIRED. |
a***e
发帖数: 1010 | 16 I agree that it may be not that important to treat RNA sample with DNaseI
during the RNA purification process in the original question. However, it is
better to maintain a "good habit" to do so.
There are several reasons for this:
(1) it doesn't add many extra work. DNaseI treatment is very simple and fast
. Qiagen has on-column DNaseI digestion kit. Or an extra TRIzol purification
step only adds one more hour.
(2) if the experiments include transfection of plasmid to overexpress some
genes or s |
n********k
发帖数: 2818 | 17 Mark! Baozi, Oncogene!
is
fast
purification
mentioned
【在 a***e 的大作中提到】
: I agree that it may be not that important to treat RNA sample with DNaseI
: during the RNA purification process in the original question. However, it is
: better to maintain a "good habit" to do so.
: There are several reasons for this:
: (1) it doesn't add many extra work. DNaseI treatment is very simple and fast
: . Qiagen has on-column DNaseI digestion kit. Or an extra TRIzol purification
: step only adds one more hour.
: (2) if the experiments include transfection of plasmid to overexpress some
: genes or s
|
