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 -70C

 

Reference:

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