Silencing DNA Repair Genes Using RNAi

RNAi oligos against EXO1 (Cat. N. SI02665138—EXO1#1 and Cat. N. SI02665145—EXO1#2), RECQ1 (Cat. N. GS5965, SMARCAL1 (Cat. N. GS50485—equimolar mixture of 4 the siRNAs) and MRE11 (Cat. N. SI02665180) were from Qiagen. Oligos were used at the final concentration of 40, 10, 20 and 20 nM, respectively. The siRNA against DNA2 was a kind gift from Dr Vindigni (14 (link)). Transfection was performed using INTERFERin (Polyplus) according to the manufacturer's instructions. As a control, a siRNA duplex directed against GFP was used. DNA2 was silenced using the pLKO-shDNA2 plasmid (Addgene #31951). The plasmid was transfected by nucleofection using the Neon system (Life Technologies). Decrease in protein levels was confirmed by western blotting at 48 h after transfection.

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Iannascoli C., Palermo V., Murfuni I., Franchitto A, & Pichierri P. (2015). The WRN exonuclease domain protects nascent strands from pathological MRE11/EXO1-dependent degradation. Nucleic Acids Research, 43(20), 9788-9803.

Publication 2015

INTERFERin Neon system

Cat 1 Cat 2 Dna2 Exo1 Neon Oligos Plasmid Protein Recq1 Rnai Sirna Transfection

Corresponding Organization :

Other organizations : Istituto Superiore di Sanità

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Variable analysis

independent variables

SiRNA against EXO1 (Cat. N. SI02665138—EXO1#1 and Cat. N. SI02665145—EXO1#2)
SiRNA against RECQ1 (Cat. N. GS5965)
SiRNA against SMARCAL1 (Cat. N. GS50485—equimolar mixture of 4 the siRNAs)
SiRNA against MRE11 (Cat. N. SI02665180)
SiRNA against DNA2 (a kind gift from Dr Vindigni)

dependent variables

Decrease in protein levels of the target genes (EXO1, RECQ1, SMARCAL1, MRE11, DNA2)

control variables

Transfection using INTERFERin (Polyplus) according to the manufacturer's instructions
SiRNA duplex directed against GFP as a control
DNA2 silencing using the pLKO-shDNA2 plasmid (Addgene #31951) transfected by nucleofection using the Neon system (Life Technologies)

controls

SiRNA duplex directed against GFP as a negative control
PLKO-shDNA2 plasmid (Addgene #31951) as a positive control for DNA2 silencing

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