Agilent tna 6000 nanokit

Snap-frozen kidney sections were recovered in RLT buffer (Qiagen) + 1% β-mercaptoethanol (Gibco) and used for mRNA extraction with Qiagen RNeasy miniKit. The quality and quantity of mRNA were evaluated with bioanalyser Agilent 2100 using Agilent TNA 6000 NanoKit and if the RNA integrity number was >7 the mRNA was retrotranscribed to cDNA. Gene markers of early tubular and endothelium activation/injury relevant for hemolysis and SCD were analyzed by low-density array (LDA, ThermoFisher) including NGAL, Kim-1, HO-1, Il-6, Ki67, ICAM-1, E-selectin and P-selectin, Caspase-3, and CD31 (16 (link), 22 (link)–25 (link)), and validated by RTqPCR for NGAL, Kim-1, HO-1, and Ki67 (ThermoFisher). Il-1β and TNF-α have been tested by RTqPCR (ThermoFisher).

Frozen kidney sections were recovered in RLT buffer (Macherey-nagel) + 1% β-mercaptoethanol (Gibco, Thermoscientific) and used for mRNA extraction using RNAse-free NucleoSpin® RNA (Macherey-nagel). The quality and quantity of mRNA were evaluated with the Agilent 2100 bioanalyzer using the Agilent TNA 6000 NanoKit (Agilent Technologies), followed by retrotranscription to cDNA (Qiagen).
Gene markers of acute tubular injury (NGAL and Kim-1), of endothelial activation (ICAM-1, VCAM-1, P-selectin, E-selectin), of inflammation (IL-6, Ki67, cNOS, Endothelin), of coagulation system (PAI-1), HO-1, and FH were analyzed by RT-qPCR (primers from ThermoFisher) using the Taqman 7900 (Life Technologies). Gene markers expression was analyzed with SDS 2.1® software (ThermoFisher), after normalization on actin housekeeping gene expression and comparison with gene expression from the pool of PBS-treated mice of each strain.