Total RNA extraction from egg and embryo cells
This method is a simple and effective way to isolate RNA from oocytes, fertilized eggs and fetal cells of frogs, sea urchins, periphyton, worms and flies.
Operation method
Total RNA extraction from egg and embryonic cells
Materials and Instruments
Triton X-100 Homogenization Buffer Phenol Chloroform Sodium Ethanol Ethanol Lithium Chloride Move I. Materials and equipment Caveat 1) Precipitation of RNA with lithium chloride facilitates the removal of glycoproteins and yolk material.2) Since the amount of RNA isolated by this method is greater than the amount of contaminating DNA, it is not necessary to remove the DNA from the product; although contaminating genomic DNA sequences do not interfere with hybridization and translation, this DNA may cause confusion if it is used in the construction of cDNA libraries, in which case the contaminating DNA is digested with tryptic DNaseI, which is an enzyme free enzyme, to remove the contaminating DNA. For more product details, please visit Aladdin Scientific website.
1)5% TritonX-100
2) homogenization buffer: 50 mmol/LNaCl, 50 mmol/LTris-Cl,(pH7.5), 5 mmol/LEDTA(pH8.0), 0.5% SDS, 200ug/ml Egg white Geminase K
3) Phenol: chloroform (1:1)
4) 3mol/L sodium acetate (pH5.2).
5) Ethanol
6)8mol/L lithium chloride
II Methods of operation
1) Place the tissue to be treated in the next dry baked glass homogenizer, discard extraneous liquids and wash the fly embryo with 0.5% TriwnX-100 to remove the medium.
2) Add 10 times the volume of homogenization buffer and grind immediately to thoroughly homogenize the tissue.
3) Warm the tissue homogenate at 37℃ for l h, with occasional mixing.
4) Transfer the homogenate to a centrifuge tube, add an equal volume of phenol: chloroform (1:1), shake vigorously for 1 min, and centrifuge at 5000 g for l0mim at room temperature with a bucket-type rotary head to separate the aqueous phase from the organic phase.
5) Transfer the upper aqueous phase to another centrifuge tube. Extract again with phenol: chloroform (1:1) according to step 4.
6) Transfer the aqueous phase into a tube, add 0.1 volume of 3mol/L sodium acetate (PH5.2), mix well, add 2.5 times the volume of ice pre-cooled ethanol, ice bath for 2 hours.
7) centrifuge at 5000 g for l5 min at 4°C, carefully remove the supernatant, and air dry the RNA precipitate at room temperature. Dissolve the RNA in a small amount of water, add 3 times the volume of ethanol to make it faster and store at -70°C. To remove glycoproteins and yolk material, perform steps 8) to 11) as required.
8) Redissolve the RNA precipitate in a small amount of water, add an equal volume of sterilized lithium chloride (8mol/L), mix well, and store at 20℃ for more than 3 hours.
9) Centrifuge the RNA at 10,000 g for 30 min at 4℃, and carefully discard the supernatant.
10) Wash the precipitate with 70% ethanol, centrifuge for a while and discard the supernatant. Dry the nucleic acid precipitate in air.
11) Redissolve the RNA in a small amount of water, add 3 times the volume of ethanol, and store at 70℃.