Ambion rna purelink kit

Murine kidney single cell suspensions were stimulated in complete RPMI with 100 ng/mL LPS (Sigma-Aldrich) for 2 hrs at 37 °C and 5% CO₂. After two washes with ice-cold PBS, cells were either stained for sorting (RNA-seq), or re-suspended in 350 μL of RTL Buffer (Qiagen, Manchester, UK) and processed with QIAshredder homogenizer (Qiagen, Manchester, UK). The follow-through was immediately frozen and stored at −80 °C for not more than two weeks before further processing.
RNA was extracted from thawed samples using Ambion RNA PureLink Kit (Life Technologies, Paisley, UK) and RNA yields analyzed by NanoDrop spectrophotometer (Thermo-Scientific, Loughborough, UK). Complementary DNA (cDNA) was prepared by using High Capacity RNA-to-cDNA™ Kit (Life Technologies, Paisley, UK) and BioRad PCR machine (BioRad, Hemel Hempstead, UK).

RNA was extracted from either whole tissue or cell suspension. Whole tissue samples were homogenised in 1 mL of RNA lysis buffer using the Precellys homogeniser system. Samples were then centrifuged at 1500xg for 10 minutes to remove contaminating material and 750 μL of supernatant taken for RNA extraction. RNA extraction was performed using the Ambion RNA PureLink kit (Life Technologies) per the manufacturer’s instructions. For expected low RNA yield the Rneasy Plus Micro kit (Qiagen) was used. RNA was quantified using NanoDrop spectrophotometer (ThermoFisher). Complementary DNA (cDNA) was prepared using SuperScript IV VILO Master Mix (Invitrogen) and amplified with BioRad PCR machine (BioRad). RT-PCR was performed using TaqMan 2× Fast Master Mix (Applied Biosystems) on the Viia 7 PCR machine (Applied Biosystems).