View source for Preparation of RNA Using Proteinase K/LiCl (Sive et al. 2000)

'''Information adapted from:'''

''Early Development of Xenopus Laevis: A Laboratory Manual.''[http://www.xenbase.org/literature/book.do?method=display&bookId=22]

2000; (First ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press. 

Sive, Hazel (Author), Grainger, Robert M (Author), and Harland, Richard M (Author).


The methods presented here have been adapted for use on Xenopus embryos. The more generally applicable methods are not provided. Although many of the protocols listed call for treatment with DEPC (diethylpyrocarbonate), this id generally not necessary when starting with good quality sterile distilled water.


'''RNA Isolation'''

Xenopus embryos contain a large amount of yolk, which can make RNA preparation from large numbers of embryos rather messy. Although only a single method is presented here, alternative methods work well as long as the volumes are increased to compensate for the yolk. This method is suitable for large numbers of embryos and can be adapted for smaller numbers by scaling the protocol appropriately. Additionally, a method for preparing RNA by in vitro transcription is given that yields RNA suitable for microinjection.

'''Preparation of RNA Using Proteinase K/LiCl'''

1. Dejelly embryos as described. [http://www.xenbase.org/xenwiki/index.php/dejellying_embryos_(Sive_et_al._2000)]


2. Allow the embryos to settle and resuspend in 4 volumes of premixed proteinase K mix.

:20 mM Tris (pH 7.6)
:100 mM NaCl
: to mM EDTA
:1% SDS
:0.5 mg/ml proteinase K


3. Homogenize the embryos by douncing with a "B" pestle. For smaller volumes (<1 ml), homogenize by vortexing.


Note: The vortexing method is inefficient for embryos beyond stage 20. They will not dissolve completely.


4. Incubate the proteinase K reaction for 1.5 hours at 37 deg C.


5. Divide the reaction into two 50-ml tubes (e.g. Corning) and add 20 ml of a 1:1 mix of phenol:chloroform to each tube.


6. Vortex the tubes and then centrifuge at 12,000xg for 5 minutes. Transfer the aqueous phases to fresh tubes.


7. Back-extract the phenol:chloroform by adding 5 ml of TE to each tube, vortexting, and centrifuging at 12,000xg for 5 minutes. Pool the aqueous phases with those obtained at step 5 above, and discard the organic phase.


8. Repeat phenol:chloroform extraction twice (without back extractions), or until there is no interface that is visible between alters, and extract once with 1 volume of chloroform.


9. Transfer the aqueous alters to fresh tubes and add 2.5 volumes of ethanol. Store for 1-2 hours at -20 deg C.


10. Collect the precipitated nucleic acids by centrifuging at 12,000xg for 20 minutes. Carefully decant the ethanol and resuspend the pellet in 360 ul of TE and 40 ul of 10x NEB4 (New England Biolabs).


11, Add 1 ul RNase-free DNase I (Ambion, 2 units/ul) and incubate for 20 minutes at room temperature.


12. Stop the DNase reaction by extracting with 800 ul of a 1:1 mix of phenol:chloroform and then with 400 ul of chloroform. Transfer the aqueous phase to a clean tube and add 135 ul of 10 M LiCl (Autoclaved). Store at 4 deg C for several hours, or overnight. LiCl will highly preferentially precipitate RNA and not DNA oligonucleotides remaining after DNase treatment.


13. Centrifuge for 20 minutes at 12,000xg. Carefully discard the supernatant and resuspend the pellet in 200-500 ul of distilled water.


14. Add 2.5 volumes of ethanols and 1.0 volume of sodium acetate (3 M, pH 4.8). Collect the precipitated RNA by centrifuging for 20 minutes at 12,000xg.


15. Redisolve the RNA in 500 ul of distilled water. Determine the concentration and quality of the solution by measuring the OD 260/280. Store the RNA as an aqueous solution at -80 deg C.


Note: Embryos contain approximately 5 ug of total RNA. This protocol routinely produces yields of 90%.


Helpful Hints:


This method can be used for smaller numbers of embryos or explants (one or two explants), but the volumes must be scaled down. Use about 50 ul of proteinase K before per embryo, or 10 ul per animal cap. Incubate the reaction for 1 hour at 37 deg C. Extract once with phenol:chloroform and treat with 1 unit of DNase I, as described above. Precipitate with LiCl (add 34 ul of 10 M LiCl for each 100 ul of RNA solution) and then with 2.5 volumes of ethanol and l1.0 volume of sodium acetate (pH 5.5).