Rna bee isolation kit

The RNA-Bee isolation kit (Tel-Test, Friendswood, TX, USA) was used according to the manufacturer’s recommendations for isolating total RNA from HaCaT cells. cDNA was prepared using reverse-transcriptase M-MuLV (Fermentas Life Science, Pittsburgh, PA, USA). PCR amplification was performed using specific primers (Bioneer, Daejeon, Korea).

Snap-frozen mouse kidneys were ground in liquid nitrogen. An RNA-Bee isolation kit (Tel-Test, Friendswood, TX, USA) was used for total RNA extraction. cDNA was obtained from the total RNA by using a first-strand cDNA synthesis kit (Transcriptor cDNA Synthesis kit, Cat. No. 04897030001; Roche Diagnostics, Mannheim, Germany). Real-time PCR was performed using an ABI ViiA7 sequence detection system (Applied Biosystems, Foster City, CA, USA) coupled with a SYBR Green or TaqMan assay (Supplementary Table S1), following the manufacturer’s instructions. For each sample, the reactions were performed in duplicate. The relative mRNA expression levels were calculated using the 2-ddCt method.

The methods used herein were modified from those described by Yang et al. (2013 (link)). To determine the abundance of Tnfxyd8 mRNA, total RNA was extracted from the whole kidney and purified using the RNA-Bee isolation kit (Tel-Test, Friendwood, TX, USA) and RNAspin Mini kit (GE Health Care, Piscataway, NJ, USA), respectively, following the manufacturer's instructions. RNA integrity was verified by 0.8% agarose gel electrophoresis. Extracted RNA samples were stored at −80°C after isolation. For reverse transcription, first-strand cDNA was synthesized using SuperScript™ Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The cDNA products were stored at −20°C until analysis via polymerase chain reaction (PCR).