Superscript 3 quantitative one step kit

Four mice randomly allocated in each group were infected with 1.0*10⁶ CFU of K. pneumoniae YJH4 or YJH15 by intravenous injection, respectively. Mice in the control group were inoculated with sterile normal saline. Blood samples were collected in each group by cardiac puncture 24hrs post-infection. Total RNA in blood was extracted and purified using the RNeasy kit (QIAGEN) and Turbo DNA free kit (Invitrogen) according to the manufacturer’s instructions. Reverse transcription analysis was conducted using SuperScript III quantitative one-step kit (Invitrogen), with GAPDH being used to normalize the expression levels of test genes. Primers used were listed in Table 2. Quantitative real-time PCR analysis was undertaken by using the QuantStudio 5 Real-Time PCR System (Applied Biosystems) and SYBR Green master mix (Applied Biosystems). Results were analyzed by QuantStudio™ Design and Analysis Software v1.5.1.

The expression level of bla_OXA-23 in strains R4-1 and R6-1 was evaluated by qPCR. Total RNA of the strains R4-1 and R6-1were extracted and purified by using the Qiagen RNeasy mini kit and Turbo DNA free kit (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was performed by using the SuperScript III quantitative one-step kit (Invitrogen), with rpoB being used as a reference gene to normalize the expression levels of test genes. Primers used in qPCR are listed in Table S1. Quantitative real-time PCR assay was performed by using the QuantStudio 7 Real-Time PCR System (Applied Biosystems) and SYBR Green master mix (Applied Biosystems). The tests were performed in triplicates. The results of qRT-PCR were analyzed by the Design & Analysis Software 2.6.0 and visualized by GraphPad prism V8.0.1.