Ask for help on RT-PCR

[yww]

Hi:
Last September I succeeded in amplifying a 1.8 Kb long viral RNA by RT-PCR=
 using 1st strand cDNA synthesis kit for RT-PCR and PCR Master (both made=
 by Boehringer Mannheim ) with the PCR machine( Perkin-Elmer) resulting in=
 the only 1.8 Kb product. But this month I can=92t repeat the result , that=
 is to say I got nothing . The only difference between these two runs is=
 the RNA sample which isolate by Promega=92s Rnagents Total RNA Isolation=
 System at different time (Just before RT-PCR each time). Can someone give=
 me some good advises on how to repeat the RT-PCR ? 

And now I still have some product got from last RT-PCR . Can I reamplify=
 the product and how to do ?

The last question is how long can I store the viral RNA isolated by=
 Promega=92s Rnagents Total RNA Isolation System under =9630 degree C in=
 Rnase freee water. (I want to reuse the RNA sample got in last September,=
 because it is now very hard to get the virus. )

Thanks in forward and waiting for your good advises eagerly .

David Yan
Yww at cims1.yzu.edu.cn