RNA extraction from schistosome intermediate hosts and parasite stages using RNAzol®

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)