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

Authors: Wannaporn Ittiprasert and Andre Miller

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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
  1. 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
  1. mRNA washes
  • Wash pellet with 0.4 ml 75% ethanol with 8,000 g centrifugation for 3 min
  • Repeat washing twice
  1. 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.

  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
  1. 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.
  1. RNA precipitation
  • Add the equal volume of isopropanol
  • Incubate RT for 15 min
  • Spin down at 12,000 g, RT, 8 min
  1. RNA washes
  • Wash pellet with 0.4 ml 75% ethanol with 4,000 g centrifugation for 3 min
  • Repeat washing twice
  1. 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

RNAzol product information

Reference:

  1. Chomczynski P. Reagents and methods for isolation of purified RNA. US Patent 7,794,932 (2010)

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Biomedical Research Institute