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
- - - - -

Increasing RNA yield from very small tissues


  • Please log in to reply
3 replies to this topic

#1 Breda3

Breda3

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 25 July 2014 - 07:54 AM

Hi,

 

I am working with very small kidney tissues from a fish(~0.5 mg or less)  and I have been having difficulties getting consistently good yields of RNA from these tissues. I started off using QIAGEN's RNeasy minikit but was unable to obtain good yields so I switched to OMEGA's MicroElute Total RNA kit which improved my yields but did not remove gDNA contamination (I had positive NORT controls during qPCR). Therefore, I treated my RNA with Ambion's DNase Treatment Set but this really reduced by RNA yields so I tried an on-column treatment instead (Qiagen's DNase Treatment set). However, the on-column treatment appears to destroy my 260/230 ratios and I also have lower RNA yields than normal. 

 

I have also tried different homogenization techniques to increase my yield including handheld homogenization, bead beating and pulse-sonication. I'd like to try grinding the tissue under liquid nitrogen with a mortar and pestle but the tissue is so small I fear I'll lose too much of it. The bead beating seems to work the best but it is still not great. I have reduced the input of RNA into my cDNA reaction which has produced successful results in qPCR but some of my tissues produce yield as low as 5 ng/uL which is lower than I'd like.

 

Does anyone have any suggestions? Pooling the samples is not an option due to limited sample size.

 

Breda



#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,826 posts
413
Excellent

Posted 27 July 2014 - 12:55 AM

You might get a better yield from a tradition extraction (i.e. phenol/chlorform or trizol) with a carrier such as linear polyacrylamide.  This would especially be the case if you are used to doing these sorts of extractions.



#3 Anh Nguyen

Anh Nguyen

    member

  • Active Members
  • Pip
  • 8 posts
0
Neutral

Posted 03 August 2014 - 04:46 AM

LPA Carrier for precipitation of small amounts of nucleic acids

The addition of linear polyacrylamide (LPA) to a nucleic acid precipitation can enhance recovery of small amounts of DNA or RNA*. LPA is an inert carrier, and is reported to not interfere with enzymatic activities, unlike some other popular carriers such as tRNA.

To an ethanol (or propanol) precipitation, add 10-20 ug of LPA

To produce a 5 ug/ul stock of linear polyacrylamide (from T. Fitzwater, ref 1, below):

In a 50 mL Falcon 2098 centrifuge tube, mix in the order listed (makes a lot, so may want to scale down to 10%):

    1. 0.25 g acrylamide (no bis-acrylamide)
    2. 4.25 mL of Millipure water
    3. 200 µL of 1 M Tris-HCl, pH 8.0
    4. 33 µL of 3 M sodium acetate, pH 7.5
    5. 10 µL of 0.5 M EDTA
    6. 50 µL of 10% APS (prepare 10% solution in sterile di-water)
    7. 5 µL of TEMED and mix well to begin polymerization; leave at room temperature 30 mins.

Add 12.5 mls 95% ETOH to precipitate approximately 5 mins.

Drain off liquid from precipitated LPA. Remove as much liquid as possible with pipet (squash pellet).

Wash pellet with 70% ETOH, then remove as much liquid as possible with pipet (squash pellet).

Air dry about 10 mins.

Add sterile di-water to LPA, let resuspend overnight with gentle shaking. Make final volume 50 mls. Concentration of LPA is 250 mg/50 mls, or 5 mg/ml, or 5 ug/ul.

Aliquot and store at -20.

Use 3 ul in a large eppy for ethanol precipitations (15 ug LPA). Be carful to not lose pellet, as it may not stick to tube as tightly as with nucleic acid carriers.

Another web protocol:

1. See LPA recipe and notes from tfitzwater (Tim Fitwater): 
http://molecularbiol... polyacrylamide

2. http://ubik.microbio...racrylamide.htm

*Gaillard, C. and Strauss, F. (1990) Nucleic Acids Res. 18, 378



#4 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,201 posts
110
Excellent

Posted 04 August 2014 - 05:34 AM

For small samples (as little as thousands of cells), we use trizol isolation with linear acrylamide carrier (commercial, from LifeTech) and increased time of precipitation steps.

The column methods have all the problem that you will get your RNA too diluted. From trizol you can disolve in any volume, i.e. 15 ul, use 1-2 ul for concentration measurement, use the rest for the DNase treatment (we use TURBO DNA Free in 20 ul reaction), and then you get out around 15 ul of treated RNA, which you can measure again and use up to 10 ul for RT.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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

©1999-2013 Protocol Online, All rights reserved.