PMC:516044 / 6996-10325 JSON TXT

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{"project":"2_test","denotations":[{"id":"15320950-11152663-12070954","span":{"begin":628,"end":630},"obj":"11152663"},{"id":"15320950-10079033-12070955","span":{"begin":1733,"end":1735},"obj":"10079033"},{"id":"15320950-10079033-12070956","span":{"begin":2809,"end":2811},"obj":"10079033"}],"text":"Quantitative RT-PCR\nAnimals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.). Kidneys and brain were removed and pulverized in liquid N2. Temporal bones were removed and two preparations of inner ear tissues were obtained by microdissection: (1) stria vascularis without spiral ligament, and (2) spiral ganglia including the surrounding bone and the organ of Corti. The dissection medium was changed twice during the microdissection and isolated tissues were washed three times to minimize cross-contamination. The Slc26a4-/-genotype was verified by the observation of large otoconia in the utricular macula [14]. Total RNA was isolated and residual DNA contamination was removed by enzymatic digestion (RNeasy micro, Qiagen, Valencia, CA). Quality and quantity of 18S rRNA obtained from kidneys and brain were determined (Nano Assay, 2100 Bioanalyzer, Agilent, Palo Alto, CA). Further, the quality of RNA preparations obtained from stria vascularis and spiral ganglia was assessed (Pico Assay, 2100 Bioanalyzer). Real time RT-PCR was performed on RNA obtained from individual animals (OneStep RT-PCR, Qiagen; Smart Cycler, Cepheid, Sunnyvale, CA) in the presence of 0.2× SYBR green I (Molecular Probes). Transcripts of 18S rRNA and mRNA for the K+ channels KCNJ10, KCNQ1 and KCNQ4 were amplified using gene-specific primers (Table 1). RT was performed for 30 min at 50°C and 15 min at 95°C. PCR consisted of 50 cycles of 1 min at 60°C, 1 min at 72°C, 7s heating to hot-measurement temperature, 13s hot-measurement at 3–5°C below product melting temperature (Table 1) and 1 min at 94°C. Hot-measurements were performed to eliminate the detection of primer-dimers that had melting temperatures between 72 and 75°C [15]. PCR was followed by a melt (60 to 95°C). The generation of a single product of appropriate size was routinely checked by the presence of a single melt peak and by agarose gel-electrophoresis. Product identity was confirmed by sequencing.\nTable 1 Primers\nTemplate Primers Product Length Melting Temperature Hot Measurement Fidelity n\n18S gag gtt cga aga cga tca ga (sense) 316 bp 83.2 ± 02°C 78°C 1.89 ± 0.02 17\ntcg ctc cac caa cta aga ac (antisense)\nKCNJ10 tgg tgt ggt gtg gta tct gg (sense) 411 bp 83.2 ± 02°C 78°C 1.86 ± 0.02 15\ntga agc agt ttg cct gtc ac (antisense)\nKCNQ1 ttt gtt cat ccc cat ctc ag (sense) 239 bp 82.5 ± 02°C 79°C 1.85 ± 0.02 17\ngtt gct ggg tag gaa gag (antisense)\nKCNQ4 ccc gga aac cct tct gtg tc (sense) 245 bp 83.2 ± 02°C 80°C 1.87 ± 0.01 17\naaa gat gag cac cag gaa cc (antisense) Template molecules (T) were quantified according to T = P / (F ^Ct) where P is the number of product molecules, F is the fidelity of the reaction and Ct is the cycle at which the number of product molecules reaches a chosen threshold [15]. Fidelity (F) was obtained from the slope (S) of the log-linear phase of the growth curve via a best-fit fifth-order polynomial: F = 7.39 + 3.80 × S + 1.05 × S2 + 0.15 × S3 + 11.38 × 10-3 * S4 + 3.39 × 10-4 × S5. The number of product molecules at threshold (PCt) was determined by amplification of known amounts of 18S rRNA according to PCt = T × F ^Ct. Quantifications of 18S rRNA were used to compare tissue amounts. Genomic contamination of inner ear samples was assessed to be \u003c0.02% by omission of the RT step."}

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PMC:516044 / 6996-10325 JSON TXT