Northern blot analysis

A premade membrane for Northern Blot from ZYAGEN company (Mouse Multiple tissue Panel NB, Cat MN-MT-2) was used to analyze Fanci expression in 22 mouse tissues. Each lane contains 20 mg of total RNA. The confirmation of quality and quantity of ribosomal RNA was provided by the company.

The membrane was first incubated in a hydridization buffer (0.5 M Sodium Phosphate pH7.2, 7% sodium dodecyl sulphate (SDS), 1 mM ethylenediaminetetraacetic acid (EDTA) pH7). Then, P³² radioactive-labeled probe covering 900 bp at the 5′ of the mouse Fanci gene was used to blot the membrane in the (same) hybridization buffer at 65°C overnight. After blotting, the membrane was washed twice 10 min in saline-sodium citrate (SSC) 2× SDS 0.5%, then twice 15 min in SSC 1× SDS 0.1% and finally twice 10 min in SSC 1× SDS 0.2%.

The 900-bp cDNA probe was generated by a touchdown PCR (from 68 to 55°C for the annealing step, then 30 cycles at 55°C) on wild-type MEFs, using JYM2632 (5′-TTTGGAAGGATCCCGAGCTG-3′) and JYM2663 (5′-AGGCATTTACTGGGATCCCCCTGCTGTCCA-3′) primers.