Authors: Wannaporn Ittiprasert and Andre Miller
Procedure
This protocol yields two RNA fractions; mRNA-containing fraction > 200 bases and microRNA-containing fraction < 200 bases.
1. Homogenization
- Homogenize the snail, parasite or related tissues using homogenizer with liquid nitrogen
- Measure the dry weight of homogenized tissues
- Add RNAzol®RT into dried pack homogenate with ratio1/1 (vol/vol)
- Mix by vortex
- DNA/protein precipitation
- Add 0.4 ml of sterile water into 1 ml homogenate
- Incubate at room temperature (RT) for 15 min
- Centrifuge at 12,000 g, RT, 15 min.
- mRNA precipitation
- Add 0.4 ml 75% ethanol into 1 ml supernatant
- Incubate RT for 10 min
- Spin down at 12,000 g, RT, 8 min
- mRNA washes
- Wash pellet with 0.4 ml 75% ethanol with 8,000 g centrifugation for 3 min
- Repeat washing twice
- RNA solubilizationwith water or Formazol®
Total RNA isolation
This protocol yields total RNA comprising all classes of RNA: large nuclear RNA, ribosomal RNA, mRNA, small RNA and microRNA down to 10 bases.
- Homogenization
- Homogenize the snail, parasite or related tissues using homogenizer with liquid nitrogen
- Measure the dry weight of homogenize powder
- Add RNAzol®RT into dried pack homogenate with ratio1/1 (vol/vol)
- Mix by vortex
- DNA/protein precipitation
- Add 0.4 ml of sterile water into 1 ml homogenate
- Incubate at room temperature (RT) for 15 min
- Centrifuge at 12,000 g, RT, 15 min.
- RNA precipitation
- Add the equal volume of isopropanol
- Incubate RT for 15 min
- Spin down at 12,000 g, RT, 8 min
- RNA washes
- Wash pellet with 0.4 ml 75% ethanol with 4,000 g centrifugation for 3 min
- Repeat washing twice
- RNA solubilizationwith nuclease free water or Formazol®
Comments
*The optional purification step using RQI (Promega), 4-bromoanisole (BAN) or other DNase enzyme can be used to further eliminate DNA contamination.
* After solubilization, the procedure for RNA must proceed on ice.
* Keep RNA at minimal temperature at -70 ○C
Reference:
- Chomczynski P. Reagents and methods for isolation of purified RNA. US Patent 7,794,932 (2010)
