Nucleospin dna kit

Total DNA was isolated to perform a polymerase chain reaction (PCR) and determine mitochondrial DNA damage. DNA was isolated from renal biopsies of eight controls subjects and twelve sepsis-AKI patients. First, sections of 5 μm thickness were cut from the renal biopsies. Samples were pretreated with 500 µL collagenase V (300 U/mL; Sigma-Aldrich, Darmstadt, Germany), incubated at 37 °C for 3 h and vortexed every 30 min. Subsequently, 500 µL RPMI (ThermoFisher, Paisly, UK) was added, followed by centrifugation for 10 min at 20,000 G at room temperature. Next, Rapid Sample Concentrator (RSC) blood DNA kit (Promega, Madison, USA) was used to isolate DNA from the pellet using Maxwell 16 MDx AS3000 (Promega), according to the manufacturer’s protocol. DNA from HUVECs was isolated with Nucleospin DNA kit (MACHEREY–NAGEL, GmbH & Co. KG, Germany), according to manufacturer’s protocol.

Genomic DNA was extracted using NucleoSpin DNA Kit (Macherey-Nagel GmbH, Germany) according to the manufacturer’s instructions, and was analyzed by the methylation-specific PCR (MSP) posterior to bisulfite conversion, as previously reported [17 (link)]. MSP primers used for OPCML promoter was as follows, M, sense: 5′-CGTTTAGTTTTTCGTGCGTTC-3′, antisense: 5′-CGAAAACGCGCAACCGACG-3′; U, sense: 5′-TTTGTTTAGTTTTTTGTGTGTTTG-3′, antisense: 5′-CAAAACAAAAACACACAACAACA-3′.

Blood samples were incubated in EDTA. Genomic DNA was extracted from peripheral blood leukocytes using the NucleoSpin DNA kit (Macherey-Nagel, Germany). PCR was performed to amplify the relevant gene region (SuperHot Master Mix, Bioron, Germany). The Ugrp2 gene has three exons; therefore, three primer pairs were used for the PCR reaction. For the first exon, 5′-GGTCAGACCGCAAAGCGAAGG-3′ was used as the forward primer and 5′-GACCTGGGATCCACGATCGG-3′ was used as the reverse primer; a 465-bp fragment was amplified. For the second exon, 5′-TGCACAGAGTTCACCGGTCCTTC-3′ was used as the forward primer and 5′-AGGGGCAGGACGGGAAACAG-3′ was used as the reverse primer; a 611-bp fragment was amplified. For the third exon, 5′-CCGCTCCCGCTCCCCACAGA-3′ was used as the forward primer and 5′-TCTCTCCCTCTCTCACGCAGCAC-3′ was used as the reverse primer; a 349-bp fragment was amplified. The NucleoFast 96 PCR kit was used for purification of the amplified products (Macherey-Nagel). Sequencing was performed for Ugrp2. The sequencing reaction was performed using purified PCR products. The obtained sequence products were purified using the ZR Sequencing Clean-up Kit D4051 (Zymo Research, USA) and prepared for array analysis. Polymorphisms in Ugrp2 were identified using the ABI PRISM 3130 Genetic Analyzer capillary automatic sequencing equipment.