RNA实验方法

Solublization of RNA in Formamide

Resuspending RNA in Formamide (as reported by Chomczynski et al. Nucleic Acids Research) has several benefits over storage in water or ethanol. First, formamide will protect RNA from RNases. The RNA is also extremely stable, allowing it to be stored overnight at 4C, or indefinitely at -20C. Samples can even be left at room temperature overnight without fear of degradation. Samples can be made to be highly concentrated, up to 4 mg/ml. Finally, with concentrated samples, no drying of your RNA is necessary for running Northerns, RT-PCR, or RNase Protection, saving time and potential degradation.

From CsCl, One Step Acid-Phenol, Trizol (tm) or RNazol B (tm) Extractions:

1. Precipitate your RNA as usual with either 100% ETOH (CsCl method) or Isopropanol (other methods). Spin to pellet.
   

2. Wash RNA pellet with 75% ETOH to remove excess salt...especially if using CsCl !!!
   

3. Aspirate ETOH, and make sure pellet is free of excess of ETOH, but DO NOT OVERDRY!!
   

4. Resuspend pellet in 100% formamide, which has been stored at 4C. Resuspend your pellet in anywhere from 10ul to 1000ul, depending upon pellet size.
   

5. Most or all of the pellet should dissolve instantly. Allow to sit at room temperature for 15 minutes. Pipet up and down frequently. If sample is very concentrated, allow to sit at 4C overnight.
   

6. To quantitate, OD 1ul in 500ul water. Remember to add 1ul of formamide to the blank as well to account for any background from the formamide.
   

7. Should the sample be too dilute, it can easily be precipitated like a water-solubilized sample, and resuspended in a smaller volume with 3M Sodium Acetate and 100% ETOH.
   

For QIAGEN RNeasy (tm) protocols:

1. After eluting the RNA from the columns with water, either dry down the sample, or precipitate with 3M Sodium Acetate and 100% ETOH.
   

2. Spin, resuspend in formamide, and OD

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