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
  • 128 posts
6
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
  • 6,676 posts
565
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
  • 1,166 posts
73
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?







Also tagged with one or more of these keywords: siRNA

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

©1999-2013 Protocol Online, All rights reserved.