Primetime qpcr assay design tool

Total RNA from adult fly heads or whole flies was extracted with the TRIzol reagent (Applied Biosystems, Grand Island, NY). First strand cDNA pool was made from total RNA (1 μg) with random hexamere oligos SuperScript II reverse transcriptase (Invitrogen, Grand Island, NY) in 20 μl reacting volume. cDNA pool was diluted (1:5) in distilled water. Gene specific assays were used to quantify genes with the SybrGreen method using the PowerSYBR Green Super PCR Mix (ABI Inc., Grand Island, NY) on an ABI7500 machine (Applied Biosystems) using default parameters. Gene specific assays were designed with the PrimeTime qPCR Assay design tool (Integrated DNA Technologies). The housekeeping gene rp49 was used as an RNA loading control as previously described (Lu et al., 2012 (link)). Data were transformed and analyzed according to the ΔΔCt method and are represented as relative fold differences (Lu et al., 2012 (link)). Primer sequences used are: sei-forward: 5′-TTATTCAAAGGCTGTACTCGGG-3′; sei-reverse: 5′-GATGCCATTCGTATAGGTCCAG-3′; ppk29-forward: 5′-CCTCTCAGGTATTCTTCGTTGG-3′; ppk29-reverse: 5′-TCGGTG-GAGATGGTATAGGTC-3′; rp49-forward: 5′-CACCAAGCACTTCATCCG-3′; rp49-reverse: 5′-TCGATCCGTAACCGATGT-3′.

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.