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In situ hybridization in zebrafish embryos frequently suffers from high background signal. Torular yeast RNA is often added to reduce the non-specific binding that leads to the high background signal. Without appropriate preparation, torular yeast RNA can also add background to the staining. This method is an easy and quick way to clean torular RNA for Hybe solution used in in situ hybridization.

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[Bio101] Preparation of Torula Yeast RNA for Hybe Solutions (Originally by Caroline Burns)

Cell Biology > Cell staining > Nucleic acid
Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
2/5/2012, 10129 views, 5 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.180

[Abstract] In situ hybridization in zebrafish embryos frequently suffers from high background signal. Torular yeast RNA is often added to reduce the non-specific binding that leads to the high background signal. Without appropriate preparation, torular yeast RNA can also add background to the staining. This method is an easy and quick way to clean torular RNA for Hybe solution used in in situ hybridization.

Materials and Reagents

  1. Torula RNA (RNA from torula yeast, type VI) (Sigma-Aldrich, catalog number: R6225, 100 g)
  2. TE (Life Technologies, Invitrogen™, catalog number: 12090-015)
  3. RNase-free H2O (Life Technologies, Ambion®, catalog number: AM9932)
  4. 3 M NaOAc
  5. Ethanol
  6. Phenol
  7. Chloroform
  8. RNase-free 80% ethanol

Equipment

  1. Standard tabletop centrifuges
  2. 50 ml Falcon tubes

Procedure

  1. Add 1 g of torula RNA powder to each 50 ml Falcon tube.
  2. Add 10 ml of phenol, then 15 ml TE to each tube.
  3. Vortex very hard until powder dissolves.
  4. After all tubes are dissolved, spin using table-top centrifuge for 10 min (spin as fast as possible).
  5. Remove top phase to a clean 50 ml Falcon tube.
  6. Do 1 phenol/chloroform extraction (1:1).
  7. Do 1 chloroform extraction.
  8. Add 1 ml 3 M NaOAc and 30 ml 200 proof ethanol to supernatent. Let stand for 5 min.
  9. Spin 20 min at full speed to pellet the RNA.
  10. Dump supernatent.
  11. Do an RNase-free 80% ethanol rinse to remove salts.
  12. Spin for 10 min.
  13. Remove supernatent and let pellet dry.
  14. Resuspend pellet in 15 ml RNase-free water.
  15. Measure the concentration on the spec (I usually do 1:100 and 1:2,000 dilution to get the OD between 0.1 and 0.9).
  16. Increase the volume to 20 mg/ml final concentration.
  17. Aliquot into 15 ml Falcon tubes and store at -20 °C.

Acknowledgments

This protocol was modified from the original protocol by Caroline Burns at the Len Zon lab, Boston Children’s Hospital, Boston, USA and supported by NIH grant R01 HL04880-21.



How to cite this protocol: Jing, L. (2012). Preparation of Torula Yeast RNA for Hybe Solutions (Originally by Caroline Burns). Bio-protocol Bio101: e180. DOI: 10.21769/BioProtoc.180; Full Text



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11/5/2012 7:52:06 AM  

What is the molar absorptivity value for Tarula Yeast RNA type IX?

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11/5/2012 7:51:31 AM  

What is the molar absorptivity value for Tarul Yeast RNA type IX?

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10/23/2012 11:52:22 PM  

What is the molar absobtivity coefficient value for this RNA?

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6/22/2012 6:22:56 AM  

I assume you are using phenol pH 4?

7/4/2012 1:38:16 PM  

Lili Jing (Author)
Department of Cell and Molecular Biology,University of Pennsylvania

Regular phenol works, like the one from invitrogen, cat #: 15513-039.

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1/21/2012 2:28:37 PM  

is ethyl alcohol or any other alcohol is used in the preparation of torula yeast?

1/28/2012 5:30:25 PM  

Lili Jing (Author)
Department of Cell and Molecular Biology,University of Pennsylvania

Yes, ethanol (ethyl alcohol, 200 proof from Pharmaco-AAPER) is used to precipitate the yeast RNA at steps 8 and 11.

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