Authors: Wannaporn Ittiprasert and Andre Miller
RNA extraction from schistosome intermediate hosts and parasite stages using RNAzol® RT
mRNA isolation
This protocol yields two RNA fractions; mRNA-containing fraction > 200 bases and micro RNA-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
2. 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.
3. 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
4. mRNA washes
– wash pellet with 0.4 ml 75% ethanol with 8,000 g centrifugation for 3 min
– repeat washing twice
5. RNA solubilization with 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 micro RNA down to 10 bases.
1. 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
2. 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.
3. RNA precipitation
– add the equal volume of isopropanol
– incubate RT for 15 min
– spin down at 12,000 g, RT, 8 min
4. RNA washes
– wash pellet with 0.4 ml 75% ethanol with 4,000 g centrifugation for 3 min
– repeat washing twice
5. RNA solubilization with nuclease free water or Formazol®
*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)
