Total RNA was isolated with TRIZOL (ThermoFisher) from a single brain hemisphere of a mixed C57BL/6 background adult mouse. 5 μg total RNA was annealed to random hexamer primers and reverse transcribed with Thermoscript (ThermoFisher) according to the manufacturer’s protocol. KCTD2, KCTD5, and KCTD17 transcripts were amplified using primer pairs oNS286 and oNS287, oNS288 and oNS289, and oNS290 and oNS291, respectively.
For in situ hybridization, DNA templates bearing a terminal SP6 promoter for in vitro transcription were generated by PCR amplification of C57BL/6 mouse genomic DNA, using primer pairs oNS1204 and oNS1205 for KCTD2, oNS1207 and oNS1208 for KCTD5, and oNS1213 and oNS1214 for KCTD17. Riboprobes were transcribed with SP6 polymerase and DIG-11-UTP or Fluorescein-12-UTP (Roche). In situ hybridization was performed as described [70 (link)], amplifying Fluorescein- and DIG-labeled probes with Fluorescein-tyramide and Cy5-tyramide (Perkin Elmer) respectively.
For in situ hybridization, DNA templates bearing a terminal SP6 promoter for in vitro transcription were generated by PCR amplification of C57BL/6 mouse genomic DNA, using primer pairs oNS1204 and oNS1205 for KCTD2, oNS1207 and oNS1208 for KCTD5, and oNS1213 and oNS1214 for KCTD17. Riboprobes were transcribed with SP6 polymerase and DIG-11-UTP or Fluorescein-12-UTP (Roche). In situ hybridization was performed as described [70 (link)], amplifying Fluorescein- and DIG-labeled probes with Fluorescein-tyramide and Cy5-tyramide (Perkin Elmer) respectively.
Free full text: Click here
