Valencia missed a chance to close within a point of Barca and Atletico when they drew 1-1 at Celta Vigo.
After Valencia goalkeeper Diego Alves had saved a 28th-minute Fabian Orellana penalty, Rodrigo fired the visitors ahead two minutes before the break before Orellana made amends with a well-taken equaliser in the 61st.
Valencia have 35 points in fourth and can be overtaken by fifth-placed Sevilla (33) if the Andalusians win at Almeria on Sunday.
Villarreal (32) and Malaga (31) stayed sixth and seventh respectively after drawing 1-1 at the latter's La Rosaleda stadium on the south coast.
Real, whose Spanish record winning streak of 22 matches was ended by Valencia, will have played one game fewer than Barca and Atletico by the end of the weekend.
Isco said Saturday's win would lift the players for Thursday's return leg at home to Atletico when they need to score at least twice to have any hope of retaining their trophy.
"It's going to be tough but we will devote all our energy to the task," he added. "With the support of the fans I think we can mount a comeback."
Reuters