Contributed by Dr.A.Gratchev

This method of first strand cDNA synthesis is more efficient than Ready-to-Go beads. You can use as little as 100ng total RNA and still detect you gene expression in PCR.

1. Bring 2 µg total RNA in the volume of 20 µl with RNase free water
2. Add 4 µl 10x DNase buffer (Ambion)
3. Add 15 µl RNase free water
4. Add 1 µl (2 U) DNase I (Ambion)
5. Incubate 30 min at 37°C
6. Inactivate DNase I for 10 min at 65°C
7. Add 5 µl Glycogen (MBI Fermentas)
8. Add 35 µl Isopropanol
9. Incubate 30 min -20°C
10. Centrifuge 20 min 14000 rpm at 4°C
11. Aspirate supernatant
12. Wash with 500 µl RNase free 70% EtOH
13. Aspirate supernatant
14. Dissolve in 10 µl RNase free water
15. Add 1 µl (0.5 µg) Oligo dT primer (Invitrogen)
16. Incubate 10 min at 70°C
17. Transfer on ice
18. Add 4 µl 5x first strand buffer (Invitrogen)
19. 2 µl 0.1 M DTT (Invitrogen)
20. 1 µl (40 U) RNase out (Invitrogen)
21. 1 µl 10 mM (2.5mM each) dNTP (Invitrogen)
22. Mix, preincubate 2 min at 42°C
23. Add 0.5µl (100U) SuperScript II (Invitrogen)
24. Incubate 50 min at 42°C
25. Freeze at -20°C

Make 1:10 and 1:100 dilutions of your cDNA and test the DNase digestion and synthesis efficiency with PDH RT-PCR.