Preparation of testis cDNA
(RNAqueous-Micro, Ambion, cat #1931)
(V. Orgogozo, 2006)
RNA extraction
1. With sterile equipment, dissect testis in cold PBS and put them in a
tube with RNAlater (Ambion, cat #7020). Keep the seminal vesicles but
remove accessory glands. Do not leave the tissues for more than a
minute in PBS. Put about 7 pairs of testis per tube. Store at 4C for up
to a month.
2. With sterile forceps, remove the testis from the RNAlater solution
and place them in a tube with 20uL of Lysis Solution. Homogenize
completely (very important, to inactivate all the RNAses present in the
tissues) with sterile blue pestles. Add 80uL of Lysis Solution.
3. Add 50uL of 100% ethanol to the lysate and vortex briefly but thoroughly.
4. Load the lysate/ethanol mixture onto a Micro Filter Cartridge Assembly and centrifuge for 10s at 13,000rpm.
5. Add 180uL of wash solution 1 and centrifuge for 10s at 13,000rpm.
6. Add 180uL of wash solution 2/3 and centrifuge for 10s at 13,000rpm.
7. Repeat with a second 180uL aliquot of wash solution 2/3.
8. Pour the flow-through. Centrifuge at 13,000rpm for 1min to remove residual fluid and dry the filter.
9. Transfer the column to a microcentrifuge tube. Apply 10uL of elution
solution, pre-heated at 75C, to the center of the filter. Wait 1min and
centrifuge for 30s.
10. Repeat with a second 10uL aliquot of pre-heated elution solution.