5.6.2.1. Full-length reverse transcription

Genomic RNA of most honey bee ssRNA viruses is approximately 10 kb long and contains highly structured 5’-terminal un-translated region with extended hairpin structures. Therefore the first strand cDNA synthesis should be performed using a reverse transcriptase with a high optimum temperature (e.g. InVitrogen’s Superscript III), so that RNA secondary structures are also transcribed.

1. Prepare purified virus particles using gradient centrifugation (see section 7; “Virus purification")
2. Extract viral RNA from the purified virus particles (see section 8.3.; “Nucleic acid extraction”)
3. Combine in a single 200 µl thin wall tube:  
3.1.  1 µg virus RNA,
3.2. 1 µl 2 µM “Reverse Primer” 5’-CGGTGTTTAAAC(T)27(X)32-3’, where (X)32 is a sequence complementary to the final 32 nucleotides at the 3’ end of the virus genome to be cloned,
3.3.  1 µl 10 mM dNTPs,  
3.4. Make the total volume 13 µl with RNase-free water.
4. Mix well by pipetting on ice.
5. Incubate at 65oC for 3 minutes in PCR heating block.
6. Transfer to ice and cool down for 1 minute.
7. Add the following:   
7.1.  4 μl 10x First Strand Buffer (supplied with Superscript III),
7.2.  1 μl 0.1M DTT,  
7.3.  1 μl RNase OUT recombinant RNAse inhibitor (Invitrogen).
8. Mix well and incubate at 52°C for 2 minutes in heating block.
9. Add 2 µl Superscript III reverse transcriptase (Invitrogen) and mix well.  

10. Incubate at 52°C for 10 min.
11. Incubate at 55°C for 60 min.
12. Incubate at 70°C for 15 min.
13. Store in freezer as a template for full length cDNA amplification.