3.3. Whole-Mount In Situ Hybridization

The colorimetric whole-mount in situ hybridization (ISH) with a PCR-amplified DIG-labeled antisense RNA probe was performed according to previously described protocols [1,15,27,28]. The synthesis of cDNA was performed using the Reverse transcriptase RNAscribe RT (Biolabmix, Novosibirsk, Russia) according to the manufacturer’s protocol. Oligo(dT) and random hexamer primers were used with a total RNA amount about 2–3 µg as a template for each reaction. DNA fragments of Mlig-SKP1 were amplified from cDNA by standard PCR with BioMaster LR HS-Taq PCR (2×), followed by cloning into the pGEM-T-Easy vector system (Promega, Madison, WI, USA) and sequenced by the SB RAS Genomics Core Facility (Novosibirsk, Russia, http://sequest.niboch.nsc.ru). Primers are listed in Table S1. DNA templates containing T7 promoter sequences at the 5′ ends of both strands for producing DIG-labelled riboprobes were amplified from the sequenced plasmid using High Fidelity Pfu polymerase (Thermo Scientific, Waltham, MA, USA). Digoxygenin-labeled RNA probe was generated using DIG RNA Labeling KIT SP6/T7 (Roche, Basel, Switzerland), following the manufacturer’s protocol. The staining was developed using NBT/BCIP colorimetric substrate (Roche). Images were made using a standard Zeiss Axio Scope.A1 inverted microscope equipped with an Axiocam 512 color (Zeiss, Jena, Germany) digital camera.