Rna labelling kit

Embryos were fixed in 4% formaldehyde using standard protocols. RNA fluorescence in situ hybridisation for CFI25 mRNA was performed as previously described [11] (link). Briefly, templates of RNA probes were obtained from PCR-amplified embryonic cDNA with the primers 5′-CGTCCAGCCGGTTAATTT-3′ and 5′- GTTAGGTAGCGCTATCGTTG-3′ (probe length of 955 bp) and cloned into pGEM-T vector (Promega). RNA probes were labelled with digoxigenin using the RNA Labelling Kit (Roche) according to the manufacturer's instructions. Fluorescent detection of RNA probes was done using anti-digoxigenin-POD (1:500, Roche) followed by Cy3 TSA amplification kit (1:50, Perkin Elmer). Antibody immunostainings were performed following standard protocols using the primary antibody rabbit anti-GFP (Life Technologies; 1:500) and secondary antibody anti-rabbit-A488 (Life Technologies; 1:750). All embryos were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei and mounted in Vectashield (Vector Laboratories). Fluorescent imaging was carried out using Leica SP8 confocal microscope. All images were processed and analysed in ImageJ.

In situ hybridization was performed as described previously.¹⁸ Briefly, cRNA probes were labelled with Digoxigenin according to RNA labelling kit (Roche). After fixation and hybridization with above probes, frozen sections were incubated with sheep anti‐Digoxigenin antibody conjugated to alkaline phosphatase (1:5000; Roche) and then stained with 5‐bromo‐4‐chloro‐3‐indolyl phosphate (BCIP, 0.4 mmol/L)/nitroblue tetrazolium (NBT, 0.4 mmol/L) buffer. The sense probe was also hybridized and served as a negative control.

The in situ hybridization probes for Nkx3.1 were generated from linearised DNA constructs containing T7 RNA polymerase recognition sites and have been previously described [13 (link)]. An RNA Labelling Kit (Roche) was used according to the manufacturer’s instructions to produce digoxigenin-labelled antisense riboprobes. Riboprobes were purified using Chromaspin 100 columns (Clontech). In situ hybridisation (ISH) was performed according to standard methods [16 (link)].

For the synthesis of the specific digoxigenin (DIG)-labeled sense and antisense RNA probes of Rgag4/Mart5, Ldoc1l/Mart6, Ldoc1/Mart7 and Cxx1a/Mart8b plasmids were linearized with restriction enzymes. In vitro transcription and DIG-labeling was performed with a RNA-Labelling Kit (Roche, Germany). All probes were then pretreated with 40 U DNaseI (Roche, Germany) for 60 min at 37°C. Tissue preparation and ISH of mouse placentae of different embryonic stages were performed according to published methods [21 (link)]. A control ISH without sense or antisense RNA probe was performed for the evaluation of artifactual background signals (Additional file 4: Figure S1).

For in situ hybridization (ISH) analyses, diethylpyrocarbonate-treated solutions were used for processing the mouse mandibles and for the hybridization experiments to ensure RNase-free conditions. We generated a 681-bp RNA probe that was complementary to the mouse FAM20A mRNA, using the same method as we previously described.^{11 (link), 24 (link)} Briefly, a primer set with 5′-ACAATTCAACCTTACCTCCTTGG-3′ (in exon 2 of the mouse Fam20A gene) for forward and 5′-CTTTTCCTGACAGCGAGTAGG-3′ (in exon 8) for reverse was used to generate a cDNA fragment using PCR, with total RNA extracted from the molars of 16.5-day-old mice as the template. Next, the PCR products were cloned into the pCRII-TOPO vector (Promega, Madison, WI, USA) and were transformed into competent Escherichia coli. The plasmid DNA was isolated from E. coli and sequenced. Digoxigenin-labelled single-stranded RNA probes were synthesized and labelled with digoxigenin using an RNA labelling kit (Roche, Indianapolis, IN, USA), and the DIG-labelled RNA probes were detected by enzyme-linked immunoassay with a specific anti-DIG-AP antibody conjugate, as we previously described.^{11 (link), 24 (link)} These RNA probes were used to detect FAM20A mRNA in the paraffin sections containing tissues from the mouse mandible.