Nucleozol one phase rna purification kit

RNA isolation for multiple tissues was achieved by using the NucleoZOL one phase RNA purification kit (Macherey-Nagel #740404.200) following the manufacturer’s instructions. Two µg of total RNA were retro-transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche Life Science, Penzberg, Upper Bavaria, Germany, #04379012001) with random hexamer primers. Real-time quantitative RT-PCR (Q-PCR) was performed in a QuantStudio 7/12k Flex System (Applied Biosystems) with the primers Crbn.F 5′-TGAAATGGAAGTTGAAGACCAAGATAG-3; Crbn.R 5′-AACTCCTCCATATCAGCTCCCAGG-3′; Hprt.F 5′-CAGTACAGCCCCAAAATGGT-3′; Hprt.R 5′-CAAGGGCATATCCAACAACA-3′; Tbp.F 5’-GGGGAGCTGTGATGTGAAGT-3′; Tbp.R 5′-CCAGGAAATAATTCTGGCTCA-3′, using the LightCycler® Multiplex DNA Master (Roche Life Science, #07339577001) and SYBR green (Roche Life Science, #4913914001). Relative expression ratio was calculated by using the 2^-ΔΔCt method with Hprt or Tbp as housekeeping genes for normalization.

RNA was isolated with the NucleoZOL one-phase RNA purification kit (Macherey-Nagel #740404.200) following the manufacturer’s instructions. An amount of 2 µg of total RNA was retro-transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche Life Science, Penzberg, Upper Bavaria, Germany, #04379012001) with random hexamer primers. Real-time quantitative RT-PCR (Q-PCR) was performed in a QuantStudio 7/12k Flex System (Applied Biosystems) using the LightCycler^® Multiplex DNA Master (Roche Life Science #07339577001) and SYBR green (Roche Life Science #4913914001). The primers used are listed in Table 1.