The Giallorossi will go through as the runners-up in Group G, after Viktoria Plzen beat CSKA Moscow earlier in the night.
With the game at the Stadio Olimpico thus becoming a straight battle for top spot, second half goals from Gareth Bale and Lucas Vazquez decided the game in the favour of the competition holders.
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The Giallorossi had their chances, especially in the first half, but Bale's strike almost immediately after the break decisively turned the tide of the game.
Roma now travel to Viktoria Plzen on Matchday 6, although only pride rests on the outcome of the game.
With Edin Dzeko ruled out through injury, Patrik Schick led the line in attack.
Nicolo Zaniolo also started, as he did in the reverse fixture at the Santiago Bernabeu.
“Besides the individual mistakes, we were missing several players today and we fielded a side with lots of youngsters. I liked our enthusiasm and aggressiveness in the first half. We did lots of good things but we need to rid ourselves of these mistakes and this fragility. Whenever we let in a goal we stop playing. That makes me furious and it's not the first time it's happened. I'm upset and angry."
- Eusebio Di Francesco
The Giallorossi face Inter Milan at the Stadio Olimpico on Sunday evening.
The sixth and final Champions League group stage game is away to Viktoria Plzen on December 12.
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