请教 RNAi knock down的检测。 - 未名空间MITBBS历史存档

国际科技财经博客移民网络热点娱乐民生时事公众号

Redian新闻

>未名空间

>Biology - 生物学

请教 RNAi knock down的检测。

请教 RNAi knock down的检测。# Biology - 生物学

a*f2010-09-01 07:09

1 楼

1
Gustavo Minas
http://klekr.com/users/[email protected]
Untitled
https://www.flickr.com/photos/[email protected]/15781838525
2
Gustavo Minas
http://klekr.com/users/[email protected]
brutas.tumblr.com
brutas.tumblr.com
https://www.flickr.com/photos/[email protected]/15594799857
3
Gustavo Minas
http://klekr.com/users/[email protected]
brutas.tumblr.com
brutas.tumblr.com
https://www.flickr.com/photos/[email protected]/15588081970
4
Gustavo Minas
http://klekr.com/users/[email protected]
Untitled
https://www.flickr.com/photos/[email protected]/15581764837
5
Gustavo Minas
http://klekr.com/users/[email protected]
http://brutas.tumblr.com/
http://brutas.tumblr.com/" rel="nofollow">brutas.tumblr.com/
https://www.flickr.com/photos/[email protected]/15145457803
6
Gustavo Minas
http://klekr.com/users/[email protected]
Untitled
https://www.flickr.com/photos/[email protected]/15571453618
For more pictures go to
http://klekr.com/editors_choice

d*n2010-09-01 07:09

2 楼

看起来很现代，用bootstrap烂大街了。
开始喜新厌旧了。可是老板不给时间re－engineer，只有望洋兴叹
另外，foundation－angular也不错，好像照jquery版的少点东西
谁有经验，给说说？

T*r2010-09-01 07:09

3 楼

针对一个基因，order了3个Thermo GIPZ lentivirus shRNAmir，侵染细胞，通过
puromycin
获得stable cell line，之后通过western blot检测，三组shRNAmir都没有明显降低蛋
白水
平。这种情况是否有必要用realtime PCR在检测一下，希望牛人能推荐一下好用的Q-
PCR kit.

i*f2010-09-01 07:09

4 楼

他不是经常拍胶片吗？

【在 a*f 的大作中提到】

: 1
: Gustavo Minas
: http://klekr.com/users/[email protected]
: Untitled
: https://www.flickr.com/photos/[email protected]/15781838525
: 2
: Gustavo Minas
: http://klekr.com/users/[email protected]
: brutas.tumblr.com
: brutas.tumblr.com

a*g2010-09-01 07:09

5 楼

I guess Q-PCR your target mRNA is worth it. (Although figuring it out does n
ot solve the Kd problem).
It's odd that three clones didn't work. Unless your gene is super essential,
and the mediocre-infected cells survived. What's the death rate after selec
tion?
Rather, I assume you have a non-infected cell line treated with same dose of
puromycin and observed complete cell death?
Some cells are just more tolerating to puromycin than others.

【在 T**********r 的大作中提到】

: 针对一个基因，order了3个Thermo GIPZ lentivirus shRNAmir，侵染细胞，通过
: puromycin
: 获得stable cell line，之后通过western blot检测，三组shRNAmir都没有明显降低蛋
: 白水
: 平。这种情况是否有必要用realtime PCR在检测一下，希望牛人能推荐一下好用的Q-
: PCR kit.

a*f2010-09-01 07:09

6 楼

过去没注意到过。

【在 i***f 的大作中提到】

: 他不是经常拍胶片吗？

T*r2010-09-01 07:09

7 楼

does n
essential,
selec
dose of
多谢答复，我所用lentivirus vector上都带有tubo GFP,所以puromycin筛选时,我拿到
是100%绿的cell line，对照组相同浓度的puromycin在一天内就可以将没转染的细胞杀
死。
我要knock down的基因是个转录因子，在发育中决定cell fate determination。
一个set中三个克隆都不起作用，很少见吗，以前做knock down比较少.

【在 a*****g 的大作中提到】

: I guess Q-PCR your target mRNA is worth it. (Although figuring it out does n
: ot solve the Kd problem).
: It's odd that three clones didn't work. Unless your gene is super essential,
: and the mediocre-infected cells survived. What's the death rate after selec
: tion?
: Rather, I assume you have a non-infected cell line treated with same dose of
: puromycin and observed complete cell death?
: Some cells are just more tolerating to puromycin than others.

m*12010-09-01 07:09

8 楼

虽然这人一直就在我的favorite里，但是我对他的片子一直不是很感冒。不过，你贴的
这几张还算我比较喜欢的。

【在 a*f 的大作中提到】

a*g2010-09-01 07:09

9 楼

then you should definetely check your target mRNA.
If possible, measure the ShRNA　products as well.
I suppose your target protein does not have an extream long half life?
WHere did you buy these ShRNA virus? The TRC (Sigma) sells some validated SH
RNA. If they fail, you can request a replacement.

【在 T**********r 的大作中提到】

:
: does n
: essential,
: selec
: dose of
: 多谢答复，我所用lentivirus vector上都带有tubo GFP,所以puromycin筛选时,我拿到
: 是100%绿的cell line，对照组相同浓度的puromycin在一天内就可以将没转染的细胞杀
: 死。
: 我要knock down的基因是个转录因子，在发育中决定cell fate determination。
: 一个set中三个克隆都不起作用，很少见吗，以前做knock down比较少.

c*a2010-09-01 07:09

10 楼

色调好看，其他的看不出好了

【在 a*f 的大作中提到】

T*r2010-09-01 07:09

11 楼

I bought the bacterial stock from Thermo Scientific Open Biosystems
Expression Arrest GIPZ Lentiviral shRNAmir. How can I check whether the
shRNA is a validated one from the sigma website? Thanks a lot!

validated SH

【在 a*****g 的大作中提到】

: then you should definetely check your target mRNA.
: If possible, measure the ShRNA　products as well.
: I suppose your target protein does not have an extream long half life?
: WHere did you buy these ShRNA virus? The TRC (Sigma) sells some validated SH
: RNA. If they fail, you can request a replacement.

s*n2010-09-01 07:09

12 楼

只有1喜欢

O*e2010-09-01 07:09

13 楼

1. Make sure your antibody really works;
2. Regular RT-PCR should be tried first to test the knock-down efficiency.

【在 T**********r 的大作中提到】

S*m2010-09-01 07:09

14 楼

我买过sigma和open biosystem的 shRNAs。 Sigma 的从来没有失败过，而open
biosystem有可能不work。我建议你从简单的步骤开始查起，先酶切看看你得到的克隆
对不对。省时间省力气。如果你拿到的是菌株，多挑几个克隆试试。

s*g2010-09-01 07:09

15 楼

你有过用SABiosciences ShRNA的经验吗?谢谢!

【在 S*******m 的大作中提到】

: 我买过sigma和open biosystem的 shRNAs。 Sigma 的从来没有失败过，而open
: biosystem有可能不work。我建议你从简单的步骤开始查起，先酶切看看你得到的克隆
: 对不对。省时间省力气。如果你拿到的是菌株，多挑几个克隆试试。

S*m2010-09-01 07:09

16 楼

没用过。不好意思，帮不上忙。我倾向于买sigma的。

【在 s*****g 的大作中提到】

: 你有过用SABiosciences ShRNA的经验吗?谢谢!

a*g2010-09-01 07:09

17 楼

search their website.
MISSION ShRNA. If it's pre-validated, they will state that so it's a selling
point.

【在 T**********r 的大作中提到】

: I bought the bacterial stock from Thermo Scientific Open Biosystems
: Expression Arrest GIPZ Lentiviral shRNAmir. How can I check whether the
: shRNA is a validated one from the sigma website? Thanks a lot!
:
: validated SH

a*g2010-09-01 07:09

18 楼

agree with you.
Bought two clones from OpenBiosystem, one of them didn't even contain any fr
agment in the vector (only if the vector was the right one)

【在 S*******m 的大作中提到】

T*r2010-09-01 07:09

19 楼

多谢大家的建议，把这几个质粒测个序先。

d*w2010-09-01 07:09

20 楼

Too expensive. We bought 5 clones from open biosystem (TRC) and two out of
them are working. The total clones only cost $300-400.

【在 s*****g 的大作中提到】

: 你有过用SABiosciences ShRNA的经验吗?谢谢!