strain: Fischer 344 gender: Male weight: 433 g developmental stage: Middle-aged tissue: RVLM
Treatment protocol
n/a
Growth protocol
n/a
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with Rneasy Lipid tissue Mini Kit followed by DNase I treatment.
Label
SYBR Green
Label protocol
Fifty ng of RNA from each sample was reverse transcribed and preamplified using an RT2 preamp cDNA synthesis kit (Qiagen, Inc.) with PBR-152Z RT2 preamp pathway primer mix (Qiagen, Inc.). Quantitative polymerase chain reactions were performed on these preamplified samples on StepOnePlus master cycler (Applied Biosystems™, Thermo Fisher Scientific) using Rat GABA & Glutamate RT2 Profiler PCR arrays (Qiagen, Inc., GeneGlobe ID PARN-152Z) with SYBR Green ROX qPCR master mix (Qiagen, Inc.,).
Hybridization protocol
n/a
Scan protocol
n/a
Description
Middle-aged RVLM Middle-aged 1504067
Data processing
Matrix non-normalized: To calculate threshold cycle (Ct) values, the background fluorescence of each well was identified by setting the start of baseline at cycle 3 and the end of baseline at cycle 15 for each array plate. The threshold value for Ct was manually selected, and the same threshold value (1.52) was used for all the arrays. After threshold selection, the Ct values of each array plate were exported as an Excel file. StepOnePlus software (Applied Biosystems) was used for Ct values extraction. Matrix normalized: The Ct values for three of the five housekeeping genes (B2m, Hprt1, and Rplp1) were identified to be consistent between arrays and were therefore used as reference genes for normalizing RNA expression. The geometric mean of the B2m, Hprt1, and Rplp1 Ct values for each experimental RVLM sample/array was used for the ΔCt value calculation of each gene. Normalization process was performed in Microsoft Excel. Genes that showed <30 Ct value in more than 50% of the arrays were used in the final analysis. Seventy-nine of the 84 GABA and glutamate neurotransmission-related genes met these criteria. In contrast, Avp, Gabra6, Gabrr1, Grm6, and Slc1a7 genes did not meet these criteria and were removed from the final analysis. Target gene signal normalized to geometric mean of B2m, Hprt1, and Rplp1 reference genes signal; 2–ΔCt, where –ΔCt = –(Ct_Target –geometric mean of B2m, Hprt1, and Rplp1 reference genes Ct values). See Data table below. Fold Change: Aged vs. young, aged vs. middle-aged, and middle-aged vs. young rats RVLM gene expression differences are represented as fold change values calculated using the 2–ΔΔCt method (Livak and Schmittgen, 2001). Fold change calculations were performed in Microsoft Excel.
Age-associated downregulation of glutamate and GABA neurotransmission-related gene expression in the rostral ventrolateral medulla of male Fischer 344 rats
