iScript cDNA Synthesis Kit

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	Follow product thermal cycling conditions

Protocol tips
Follow product thermal cycling conditions

Publication protocol

cDNA synthesis was performed using the iScript™ cDNA Synthesis Kit (BioRad), with 10 ng or 1 μg of total RNA or 10 ng of rRNA-depleted RNA (processed with MICROBExpress, RiboMinus, or Ribo-Zero kit) used as the template. Subsequently, qPCR for indicated transcripts was performed using the BioRad CFX Connect Real-Time PCR Detection System and SsoAdvanced™ SYBR® Green Supermix (BioRad) with oligonucleotides listed in Table 1. In order to establish standard curves for rRNA, mreB, brlR, and PA0701 transcript copy numbers versus Cq values, as calculated using the CFX Manager Software (BioRad), qPCR was also performed using serial 10-fold dilutions of P. aeruginosa genomic DNA. Subsequently, the standard curves were used for calculations of template copy numbers in the indicated samples. Melting curve analyses were employed to verify specific single product amplification. The qRT-PCR analysis was performed using biological triplicates.

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Manufacturer protocol

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