Quantitative PCR analysis

The lnc SINEUP expression in the isolated clones (pool, 1, 3, 12, 15) was evaluated by using quantitative RT-PCR. Cells were lysed by TriFast (EuroClone) and the total RNA was extracted with phenol/chloroform. 5 µg of RNA were treated with RQ1 RNase-free DNase (Promega) to eliminate residual DNA contaminants. After DNase inactivation, 1 µg of RNA was reverse-transcribed by using ImProm-II Reverse Transcriptase (Promega) in a mix containing 3 mM MgCl₂, 0.5mM dNTP and 500 ng random primer examer mixture (Invitrogen). The cDNA was then amplified in a 7500 Real-Time PCR System (Applied Biosystem) using SYBR Green PCR Mastermix (Applied Biosystem). All oligonucleotide primers (listed below) were used to a final concentration of 0.2 µM.

ACTB FOR: 5′-CGTGCTGCTGACCGAGG-3′

ACTB REV: 5′-GAAGGTCTCAAACATGATCTGGGT-3′

SINEUP_FOR: 5′-TCCTGTGCAAGAGCATCCAG-3′

SINEUP_REV: 5′-TCCCTTGCTGTTCGTTCGTT-3′

The relative abundance of target RNAs was evaluated in relation to β-actin (ACTB) transcript by ΔΔCt method. ²¹ Correlation analysis between mAbs productivity and SINEUP expression was assessed by dispersion analysis and Pearson's correlation index.