Special Feature

User Panel

My Panel

My Panel

Bookmark Science Articles

Recent News
Bookmark / Share This Science Site

RNA Isolation

RNA Extraction Background Information

Obtaining high quality, intact RNA is the first and often the most critical step in performing many fundamental molecular biology experiments, including Northern analysis, nuclease protection assays, RT-PCR, RNA mapping, in vitro translation and cDNA library construction. To be successful, however, the RNA isolation procedure should include some important steps both before and after the actual RNA purification.

Basic RNA Isolation Methods

Depending on the RNA to be extracted, there are properties of RNA that allow it to be isolated away from other cellular materials such as proteins, DNA and even lipids.

Messenger RNA or mRNA contains a stretch adenosine residues in the form of a poly(A) tail. This has been exploited to allow mRNA to be bound to poly(T) affinity columns or beads.

Tips for RNA Extraction and Isolation

If you currently have a preferred method for isolating RNA, continue to use it.

If you haven't isolated RNA from your system before and your organism is not on the following list, we can help you identify established protocols that have been used for isolating RNA for Northern blotting or RT-PCR from your system. These types of protocols will also yield "microarray-grade" RNA.

Always run an agarose gel of your RNA to assess the quality.

NOTE: If you use a non column based purification reagent such as TRIzol we recommend you precipitate your RNA a second time from ethanol or you pass it through an Rneasy column or similar device. This insures removal of all organic contaminants (See spectra below).

 

RNase Inhibitors Used in RNA Extraction and Isolation

 

Checking RNA Isolation Quality and Quantity

Efficient nuclear/cytoplasmic fractionation can be quickly assessed by denaturing agarose gel analysis (see Figure 1). Bands correspond-ing to precursor rRNA should be equivalent in the total and nuclear RNA fraction. The 18S and 28S rRNA bands will be less intense in the nuclear RNA fraction than in either total RNA or cytoplasmic fraction RNA. In some cell lines, for example 293 and COS-7 cells, this difference will be dramatic, but in other cells lines such as HeLa cells, the rRNA bands are only slightly less intense in nuclear RNA than in total or cytoplasmic fraction RNA."

RNA Purity

A260 readings and 260/280 ratios are not enough to ensure high purity RNA. You must take a full UV spectrum. Below are 3 example spectra which all have good 260/280 ratios, but have very different purities. You can also use the 260/230 ratio to help determine the amount of organic contamination in your RNA. It is a much more variable number than the 260/280 ratio, but generally, ratios above 1 indicate good purity.

 

Quantifying RNA Extraction and Isolation

 

RNA Isolation from Subcellular Fractions

Cells were then washed in PBS and successively extracted as described. First, cells were extracted with the Nonidet P-40 buffer (10 mM Tris-Cl, (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.5% (v/v) Nonidet P-40, 40 units/ml Rnasin, 1 mM DTT). The remaining pellet (rough endoplasmic reticulum (RER) and nucleus) was washed in the same buffer and extracted in buffer B (10 mM Tris-Cl (pH 7.5), 10 mM NaCl, 0.5%(v/v) Nonidet P-40, 40 mM EDTA, 40 units/ml Rnasin, 1 mM DTT). The remaining nuclear pellet was washed in the same buffer and extracted with high salt buffer (10 mM Tris-Cl (pH 7.5), 0.5 M NaCl, 3 mM MgCl2, 0.5%(v/v) Nonidet P-40, 40 units/ml Rnasin, 1 mM DTT). RNA was purified from each fraction by the RNA STAT solution. Total RNA was isolated using the RNA-STAT60 reagent (Tel-test) according to the instructions provided by the manufacturer. (Reference: Byeong-Churl Jang et al., 2002)

 

RNA Isolation Using CsCl Protocol

Chaos Buffer for RNA isolation

For 100 mls 200 mls

4. 5M Guanidinium thiocyanate 53.2 g 106.4 2% N-lauroylsarcosine (sarcosyl) 2.0g 4.0g 50mM EDTA 0.5M 10 mls 20 mls 25mM Tris-HCl, pH 7.5 1M 2.5 mls 5 mls

0. 1M b-mercaptoethanol 700ul 1.4mls

0. 2% antifoam A (Sigma) 200 ul 400 ul

5. 7 M CsCl cushion* Final Volume 100 ml

5. 7 M "Baked" CsCl 95.8g

0. 1M EDTA 0.5M 20 mls

RNA Extraction Procedure Using CsCl

* Use DEPC treated water and baked glassware. This solution, absolutely, positively must be Rnase free. Rnase is everywhere! Wear gloves, use only baked glassware, or virgin plastic. DePC treat your water, buffers (except Tris). Be very careful.

1. Weigh animal. Remove organ (liver), weight One of the superior attributes of cDNA is you reduce the gene complexity by choosing an organ that only expresses a single locus of a multilocus enzyme. 2 . Homogenize tissue rapidly in "Chaos" buffer (9mls) 3 . Spin homogenant at 12 KG to remove insoluble material 4 . WHILE Homogenant IS SPINNING 4A. Weight out 1.8 g of CsCl (0.2g/ml) per sample. 4B. In the ultra-centrifuge tube ("quick-seal") add 3.5mls of 5.7M CsCl "cushion" This layer must be Rnase free. You will centrifuge your RNA through this layer and any contamination will cause significant losses. 5 . Remove supernant, put into fresh tube, add 1.8 g CsCl 6 . Carefully, add homogenant on top of the CsCl cushion. This is most readily accomplished by using a 10cc syringe and long needle. Place needle/syringe into quick-seal tube, and add tissue homogenant to the syringe. Homogenant will slowly trickle into Q-S tube, floating on top of the cushion.

7. Carefully, balance matched pairs of centrifuge tube (+/-0.005 g).

8. Seal tubes, place matched tubes opposite of each other and cap with red aluminum tops.

9. Centrifuge, 50,000 rpms 5.5 hrs, 20oC

After centrifugation: Mark tubes where pellet should be: on the bottom outer side (relative to center of rotor)

10. Remove tubes, place in convenient rack.

11. Slice off the top of tube.Aspirate off the top 2/3- 3/4 of solution. WORK FROM THE TOP DOWN. IT IS IMPORTANT THAT YOU ASPIRATE OFF THE UPPER MOST SOLUTION FIRST AND REMOVE ALL BUT THE CLEAR CUSHION. DO NOT ASPIRATE THE INVISIBLE PELLET AT THE BOTTOM.

12. Cut off the top 2/3 of tube. Using a "pulled" Pasteur pipet, carefully remove remaining solution. The pellet will be on the bottom-side of the tube.

13. Rinse pellet with 70% EtOH, remove (use Pasteur pipets), repeat twice (total 3 washes)

14. Add 200 ul of 0.1x TE (RNase free), using the pipet tip to crush pellet and "titrating" T.E. attempt to put pellet into solution. At least form a heterogeneous solution and put into a fresh microfuge tube.

15. Repeat with 200 ul of T.E. pooling both solutions

16. Vortex, good RNA does not like to go into solution. Patience is a virtue.

17. Determine concentration, 10 ul into 490 ul (500 ul final vol)

18. Remove 1 to 2 ug of RNA for gel analysis (add RNA loading buffers)

19. Add 1/10 volume Rnase free 3M NaOAC (40 ul), 2.5 volumes of EtOH (1.0ml). Mix vigorously.

20. You can calculate the concentration of RNA in the EtOH.Thus, there is no need to precipitate all of your RNA at once. Just remove about 1.5 to 2 times the amount you need by vortexing EtOH/RNA solution and remove the appropriate volume. This EtOH/RNA solution stored at 20oC or lower, is stable for years.

See:

RNA Protocols

RNA Bioinformatics

 

Science News

For science news click here:Science News