Smartscribe reverse transcriptase enzymes

Total RNA was isolated from fine dissected OC isolated from P10 inner ear tissue of wild-type, heterozygous and Map3k1^tm1Yxia homozygous mice (five mice each) using the RiboPure RNA isolation kit (Life Technologies, Grand Island, NY) and cDNA was prepared using an oligo-dT primer and SMARTScribe Reverse Transcriptase enzymes (Clontech, Mountain View, CA). To determine the differential expression of various genes, SYBR-Green-based real-time primers (available upon request) were designed using Integrated DNA Technologies online PrimeTime qPCR assay design tool (http://www.idtdna.com/Scitools/Applications/RealTimePCR/). The real-time PCR assays were performed in triplicate using an ABI StepOnePlus Real-Time thermalcycler (Life Technologies, Grand Island, NY). C_T values were normalized using Gapdh and actin as an endogenous control, and fold changes of different genes were calculated using SABiosciences online software (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). The genes with a twofold change and with a P-value less than 0.05 based on a Student's t-test analysis were considered significant.

The whole inner ear was isolated from P150 WT and Ppip5k2^K^^/K^ mice using the RiboPure RNA isolation kit (Life Technologies, Grand Island, NY). cDNA was prepared using an oligo-dT primer and SMARTScribe Reverse Transcriptase enzymes (Clontech, Mountain View, CA). To determine the differential expression of Ppip5k1, SYBRGreen based real-time primers were designed using Integrated DNA Technologies online PrimeTime qPCR assay design tool. The real-time PCR assays were performed in triplicate using an ABI StepOnePlus Real-Time thermal cycler (ABI, Foster City, CA). CT values were normalized using Gapdh as an endogenous control, and fold changes of Ppip5k1 transcripts in different tissues were calculated using 2^-ΔΔCt standard formula. An expression with a 2-fold change and with a P-value less than 0.05 based on a Student’s t-test analysis was considered significant.