s********9
发帖数: 132 | 1 Evidence for isoforms:
1: western blot shows two bands.
2: both bands sensitive to siRNA-1, only one band sensitive to siRNA-2.
the isoforms listed in Genebank do not explain these observations, which
suggests a new isoform. What would be the routine experiment to identify
this new isoform?
Thanks a lot. |
T*****e
发帖数: 247 | 2 坐等大神解答。
如果你有一个抗体只能识别siRNA-2的目标区，并且用这个抗体做WB只剩一条带。我觉
得我会更相信一些。
说的不对请见谅。 |
l***y
发帖数: 638 | 3 做全长mRNA的RT，或者单独把siRNA2的binding区RT-PCR弄出来去测序
话说不是啥重要蛋白的isoform有人关心吗？
【在 s********9 的大作中提到】
: Evidence for isoforms:
: 1: western blot shows two bands.
: 2: both bands sensitive to siRNA-1, only one band sensitive to siRNA-2.
: the isoforms listed in Genebank do not explain these observations, which
: suggests a new isoform. What would be the routine experiment to identify
: this new isoform?
: Thanks a lot.
|
s********9
发帖数: 132 | 4 The reported mRNA isoform should be targeted by both siRNAs.
The unreported one is insensitive to siRNA-2. I should've made it clear.
【在 l***y 的大作中提到】
: 做全长mRNA的RT，或者单独把siRNA2的binding区RT-PCR弄出来去测序
: 话说不是啥重要蛋白的isoform有人关心吗？
|
z***k
发帖数: 33 | |
s********9
发帖数: 132 | 6 Evidence for isoforms:
1: western blot shows two bands.
2: both bands sensitive to siRNA-1, only one band sensitive to siRNA-2.
the isoforms listed in Genebank do not explain these observations, which
suggests a new isoform. What would be the routine experiment to identify
this new isoform?
Thanks a lot. |
T*****e
发帖数: 247 | 7 坐等大神解答。
如果你有一个抗体只能识别siRNA-2的目标区，并且用这个抗体做WB只剩一条带。我觉
得我会更相信一些。
说的不对请见谅。 |
l***y
发帖数: 638 | 8 做全长mRNA的RT，或者单独把siRNA2的binding区RT-PCR弄出来去测序
话说不是啥重要蛋白的isoform有人关心吗？
【在 s********9 的大作中提到】
: Evidence for isoforms:
: 1: western blot shows two bands.
: 2: both bands sensitive to siRNA-1, only one band sensitive to siRNA-2.
: the isoforms listed in Genebank do not explain these observations, which
: suggests a new isoform. What would be the routine experiment to identify
: this new isoform?
: Thanks a lot.
|
s********9
发帖数: 132 | 9 The reported mRNA isoform should be targeted by both siRNAs.
The unreported one is insensitive to siRNA-2. I should've made it clear.
【在 l***y 的大作中提到】
: 做全长mRNA的RT，或者单独把siRNA2的binding区RT-PCR弄出来去测序
: 话说不是啥重要蛋白的isoform有人关心吗？
|
z***k
发帖数: 33 | |
n******7
发帖数: 12463 | 11 ...大炮打蚊子
做个sanger就好了
【在 z***k 的大作中提到】
: targeted RNA-seq.
|
n******7
发帖数: 12463 | 12 可以预测一下siRNA-2的target site
然后看在哪个exon上
可能是exon skipping的事件
这是最简单的情况
复杂的就很复杂了
你可以测序验证
如果是有生物功能的isoform的话 还是有点意思的
【在 s********9 的大作中提到】
: The reported mRNA isoform should be targeted by both siRNAs.
: The unreported one is insensitive to siRNA-2. I should've made it clear.
|
s********9
发帖数: 132 | 13 I know the sequence of both siRNA.
I want to clone this unrecognized isoform and express it to study its
function.
BTW, how do I do sanger seq to get the seq of unrecognized siRNA-insensitive
isofrm? (product is smaller than all reported isoforms)
And can Mass Spectrometry identify the sequence of this unrecognized isoform
(let's call short form)?
WHat i worry about is the contamination from reported isoform (let's call
full length)
【在 n******7 的大作中提到】
: 可以预测一下siRNA-2的target site
: 然后看在哪个exon上
: 可能是exon skipping的事件
: 这是最简单的情况
: 复杂的就很复杂了
: 你可以测序验证
: 如果是有生物功能的isoform的话 还是有点意思的
|
n******7
发帖数: 12463 | 14 1. 最好先根据siRNA的靶点估测一下这个isoform的序列构成
在某个coding exon上面最好
2. 如果大致确定是alternative splicing，而不是5'-/3'-的变化
可以根据全长isoform 的ORF设计引物做RT-PCR
如果这个short-form 表达量不是特别低的话
做一定数量的clone就可以抓住了
后续你怎么玩都可以了
3. 可以同时做一些bioinfo的分析
比如这个AS event很可能对应新的splicing junction
可以看一下已有的RNA-Seq data里面这个junction的表达情况
由此可以大致推断这个isoform是不是functional的
另外，也可以居然protein sequence的变化，看看是不是有些domain被打乱了
这样的话有可能跟全长蛋白功能相反的，这是比较有意思的一种情况
insensitive
isoform
【在 s********9 的大作中提到】
: I know the sequence of both siRNA.
: I want to clone this unrecognized isoform and express it to study its
: function.
: BTW, how do I do sanger seq to get the seq of unrecognized siRNA-insensitive
: isofrm? (product is smaller than all reported isoforms)
: And can Mass Spectrometry identify the sequence of this unrecognized isoform
: (let's call short form)?
: WHat i worry about is the contamination from reported isoform (let's call
: full length)
|
