s****6
发帖数: 34 | 1 Hello, Friends, Would you please share your experience in handling those
hypothetical genes in microarray data? If they could be confirmed by Q-PCR,
they are valid discovery. But what is the best strategy to proceed. Do you
ignor them or love them better than others because of a sure novelty here?
Thanks a lot for your inputs. |
t*d
发帖数: 1290 | 2 我现在在做这个，也在摸索。所以随便讲讲。
我们现在做的是比较treatment以后，基因表达的变化。microarray找出了好几百个基
因。所以我先用 GSEA 做了一个pathway/geneset analysis，看看那个pathway/
geneset 显著的变化了。然后从几个感兴趣，又显著的pathway/geneset中，找出那些
表达变化比较大的基因，用q－PCR，western，immunohistochemistry验证。然后和已
经发表的文献联系联系，扯上几句。比如A基因和细胞增殖有关，我的处理也和增殖有
关，但是A基因和我的处理的关系没有被报道，这样我在discussion里讨论讨论，把他
们联系起来。当然，如果条件许可，进一步做一些 gain of function 和 loss of
function 的实验，就更好了。做 GOF 和 LOF 比较费时，所以多看看文献，再决定哪
个基因的变化更可能有表型。这个时候你自己的 microarray 数据帮不上太多的忙，需
要你的专业知识，别人的报道，别人的 microarray 数据 （比如有人做过的有临床数
据 |
s****6
发帖数: 34 | 3 Thank you very much for this wonderful sharing. While what you said is very
valuable and I appreciate it, I was asking about how to deal with those
undefined or uncharacterized genes, like those we met when we used the Affy
HG-U133_plus 2 chips. Some of the probes are from EST and unknow genes. I
got a few of them significantly changed in my treatment. Is it worthwhile to
pursue those "genes" for the sake of novelty? If yes, how to proceed? Do I
have a burden to prove these are truely some gene
【在 t*d 的大作中提到】
: 我现在在做这个，也在摸索。所以随便讲讲。
: 我们现在做的是比较treatment以后，基因表达的变化。microarray找出了好几百个基
: 因。所以我先用 GSEA 做了一个pathway/geneset analysis，看看那个pathway/
: geneset 显著的变化了。然后从几个感兴趣，又显著的pathway/geneset中，找出那些
: 表达变化比较大的基因，用q－PCR，western，immunohistochemistry验证。然后和已
: 经发表的文献联系联系，扯上几句。比如A基因和细胞增殖有关，我的处理也和增殖有
: 关，但是A基因和我的处理的关系没有被报道，这样我在discussion里讨论讨论，把他
: 们联系起来。当然，如果条件许可，进一步做一些 gain of function 和 loss of
: function 的实验，就更好了。做 GOF 和 LOF 比较费时，所以多看看文献，再决定哪
: 个基因的变化更可能有表型。这个时候你自己的 microarray 数据帮不上太多的忙，需
|
t*d
发帖数: 1290 | 4 抱歉，误解了你的意思。还扬扬傻傻写了一大堆，呵呵。
这种 unknown 基因我一般是不去碰的。为什么呢？因为microarray结果的假阳性太高
了。就算你 rank 后，排在第一那个的都有可能是个假阳性。所以在做任何后续实验，
甚至简单的validation（用qPCR等方法）前，都最好综合各方面的知识，给candidate
genes排个序。然后决定试哪个。而这些未知的基因，后面要做的实验太多了。最难的
是找到它表达的蛋白，找到它的蛋白的抗体。够你折腾老长一段时间了。
当然，你有其它的证据。比如你发现在某个 GWAS 中，这个基因的 SNP 和某个疾病极
强相关，然后在你的与这个疾病相关的处理中表达变化了，然后qPCR验证了这个基因的
表达变化。这时也许值得一试。
very
Affy
to
I
【在 s****6 的大作中提到】
: Thank you very much for this wonderful sharing. While what you said is very
: valuable and I appreciate it, I was asking about how to deal with those
: undefined or uncharacterized genes, like those we met when we used the Affy
: HG-U133_plus 2 chips. Some of the probes are from EST and unknow genes. I
: got a few of them significantly changed in my treatment. Is it worthwhile to
: pursue those "genes" for the sake of novelty? If yes, how to proceed? Do I
: have a burden to prove these are truely some gene
|
s****6
发帖数: 34 | 5 Great. That is helpful. Thanks a lot. |
a***e
发帖数: 1010 | 6 it is hard to analyze those unknown genes. However, new biology always
comes out something novel and unknown. |
l******n
发帖数: 105 | 7 高手啊，讲得太漂亮了！版主应该发奖励吧。
【在 t*d 的大作中提到】
: 我现在在做这个，也在摸索。所以随便讲讲。
: 我们现在做的是比较treatment以后，基因表达的变化。microarray找出了好几百个基
: 因。所以我先用 GSEA 做了一个pathway/geneset analysis，看看那个pathway/
: geneset 显著的变化了。然后从几个感兴趣，又显著的pathway/geneset中，找出那些
: 表达变化比较大的基因，用q－PCR，western，immunohistochemistry验证。然后和已
: 经发表的文献联系联系，扯上几句。比如A基因和细胞增殖有关，我的处理也和增殖有
: 关，但是A基因和我的处理的关系没有被报道，这样我在discussion里讨论讨论，把他
: 们联系起来。当然，如果条件许可，进一步做一些 gain of function 和 loss of
: function 的实验，就更好了。做 GOF 和 LOF 比较费时，所以多看看文献，再决定哪
: 个基因的变化更可能有表型。这个时候你自己的 microarray 数据帮不上太多的忙，需
|
s****6
发帖数: 34 | 8 any more input on this thread? please, thanks. |
a***e
发帖数: 1010 | 9 if only for publication, don't touch them.
for novelty issue, I like them more and want to spend more time on that. |
s****6
发帖数: 34 | 10
hypothetical genes in microarray data? If they could be confirmed by Q-PCR,
they are valid discovery. They are new for sure in terms of their function
in your questions. And therefore worthwhile for further study. But what is
the best strategy to proceed. Do you ignor them or love them better than
others because of a sure novelty here? Thanks a lot for your inputs.
【在 s****6 的大作中提到】
: Hello, Friends, Would you please share your experience in handling those
: hypothetical genes in microarray data? If they could be confirmed by Q-PCR,
: they are valid discovery. But what is the best strategy to proceed. Do you
: ignor them or love them better than others because of a sure novelty here?
: Thanks a lot for your inputs.
|
