Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo

A help for siRNA experiment

siRNA

  • Please log in to reply
2 replies to this topic

#1 madelingirly

madelingirly

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 113 posts
4
Neutral

Posted 04 February 2014 - 11:13 PM

Dear All,

at first, I am still a beginner in siRNA experiments, this is my first one

I want to silence specific two genes

I bought siRNA from sante cruz company.

http://www.scbt.com/

with all kit, like transfecting agent, medium and control siRNA.

and I cultured my cells and followed the protocol as written exactly,

after finishing the experiment and waiting 24 hrs, I wanted to test the presence of this protein in the cell i silenced, so I stained my cells by immunofluorescnece.

However, I could not see any difference, between, control siRNA and gene silenced and even normal cells.

so I tried again, with 2 experiment, 1 changing the transfection reagent into RNAi fect, and 2- repeat the same old experiment but waited for both exp longer time, perhaps my protein half life time is long (65 hrs)

however, today, I can see the same under microscope.

for both genes I used.

 I know that I need rt-PCR or western blot to prove silencing of specific gene.

 

my questions?

The use of immunofluorescence staining is good way to prove silence gene?

Do u have any suggestion for my to think about since I am beginner?

Do u have any experience regarding this company and siRNA products

 

at the end, I am very grateful for those who is welling to share their experience with me

Thank u

 

 



#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,800 posts
407
Excellent

Posted 05 February 2014 - 01:13 AM

I have two comments - does this antibody work for you with IF in other experiments?

 

and

 

I would start with looking at KD using western blot - that is the functional end of things.  Yes, IF is similar, but the major difference is that IF i mostly not a very quantitative technique, whereas westerns can be.  So, if you KD and only see a 50% reduction in the WB, you might not be able to visualize this on IF.



#3 pcrman

pcrman

    Epigenetist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,165 posts
68
Excellent

Posted 05 February 2014 - 01:15 AM

Hi madelingirly,

 

here is a list of things you need to think about when trouble shooting:

1) Transfection efficiency: how well have the siRNAs been transfected into your cells? In general, siRNA transfection efficiency is not a big concern, but may vary in different cells. How about you purchase a fluorescence labeled siRNA as a control for transfection efficiency?

2) I don't think IF is a good way of accessing gene knockdown. IF itself can give you false positive signal. Try RT-PCR and western.

3) Do you know the basal expression of the target genes in your cells? Are they already expressed low?







Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.