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
- - - - -

Placental RNA extraction with qiagen rneasy mini plus kit


  • Please log in to reply
9 replies to this topic

#1 sheon

sheon

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 05 March 2013 - 05:59 AM

Hello,

I am trying to extracted RNA from human placenta using RNeasy mini plus kit. The protocol followed was as mentioned by the user manual and when sufficient yield was not obtained, some variation in protocol was made. However, I am getting continousely very low yield. The initial tissue taken was 30 mg from which I obtained 100ng/ul concentration (nanodrop reading), RNA elution was done by 15ul Rnase free water and A260/280 was 2.01.

I want following suggestion:

1. What should I do to obtain more yield?
2. How much yield will you get normally from placental tissue?


Please Help

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,505 posts
370
Excellent

Posted 05 March 2013 - 11:42 AM

Are you using the tissue fresh or is it being stored (and how is it being stored)? Have you looked at the RNA on a gel?

#3 sheon

sheon

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 05 March 2013 - 08:26 PM

Tissue was stored in -80 degree deep freezer.
Yes, i have seen the RNA on gel. It shows 28S and 18S bands and a smear starting from just above the 28S till below of 18S. The quantity on gel also looks very less.

#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,505 posts
370
Excellent

Posted 06 March 2013 - 01:06 PM

OK, it sounds like the DNA may be partly degraded from the smear; for intact RNA you should only see the rRNA bands and maybe a low MW smear (mRNA) if you loaded enough. It may be that the tissue hasn't been stored properly or wasn't frozen fast enough when harvested. Unfortunately there isn't much you can do about this, other than to get fresh samples. However, the RNA should still be useable.

As to the yield issues - have you tried a Trizol (phenol:chloroform) extraction or another kit? Typically it is not a good idea to modify the kit procedures, they have been optimised for the kit. Perhaps using 30 mg is too much for one of the steps (probably the digestion/disruption step), have you tried to use less tissue in an extraction?

#5 sheon

sheon

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 06 March 2013 - 08:23 PM

Yes, I have tried the trizol method, in which i had used 100mg tissue and got the yeild of 1000ng/ul, however the purity of RNA was in range of 1.6 to 1.8, and the pattern of gel image looked similar to the one which i am getting with kit method.

My concern is the sample amount which I get is very less. With trizol method I am losing a lot of tissue which I cant afford, plus the purity is not the same how i get with kit.

I have tried using low amount of tissue (5,10 and 20 mg) also for the kit method, but the results i get on gel are the same. Following are the experiments i did: Attached File  Sample.doc   39.5KB   147 downloads

Edited by sheon, 06 March 2013 - 08:30 PM.


#6 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,505 posts
370
Excellent

Posted 06 March 2013 - 11:11 PM

Hmmm. Do you have access to another tissue (or cells) that you could extract to see if it is something you are doing wrong or something with the tissue? Your experiments look pretty convincing. There may be something about placental tissue that gives low RNA yield.

#7 sheon

sheon

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 06 March 2013 - 11:40 PM

ok. I will try to see if I can get access to other tissue (though it will be difficult, since my lab mostly works on plant system).

I wanted to know how much is normally the RNA YIELD from placental tissue.

#8 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,505 posts
370
Excellent

Posted 07 March 2013 - 12:02 PM

No idea sorry, but in general a kit like that with 30 mg of tissue should give you about 25 ug of RNA, though it is quite tissue dependent.

#9 sheon

sheon

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 10 March 2013 - 12:14 AM

Hello Bob,

I tried combining trizol method and kit method together. I am getting a yield of around 800ng/ul from 30mg tissue with purity A280/260 of 2.01.

#10 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,505 posts
370
Excellent

Posted 10 March 2013 - 11:42 AM

Cool, sounds like you have a high level of RNases present in the placenta (makes sense to me, it has a vital function to filter blood for the baby and parent, similar to liver I guess), so the trizol inhibits the RNases and the kit does the rest.




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

©1999-2013 Protocol Online, All rights reserved.