Revertaid h minus rt kit

Intracellular RNA was isolated using RNA-Solv Reagent (Omego Bio-Tek, #R6830) and reverse transcribed into complementary DNA, applying the RevertAid H minus RT Kit (Thermo Scientific, #EP0452). Subsequently, quantitative real-time PCR for specific transcripts was carried out with the Maxima SYBR-Green qPCR Kit (Thermo Scientific, #K0221). The housekeeping gene hRPL27 was used as internal control. All gene-specific primers used in this study are listed in Table 2. Samples were measured in duplicate with the LightCycler 480 Instrument II (Roche) and analyzed as fold-change values as compared to the control, achieved by applying the ΔΔC_T method (52 (link)).

The expression of full-length pituitary GHRH receptor (pGHRH-R) and GHRH was detected by RT-PCR. Total RNA was isolated using NucleoSpin kit (Macherey-Nagel Inc., Bethlehem, PA). The yield and quality of total RNA was determined spectrophotometrically using 260 nm and 260/280 nm ratio, respectively. Genomic DNA was eliminated using Turbo DNA-free kit (Life Technologies, Carlsbad, CA). One and a half μg of RNA was reverse transcribed into cDNA with the RevertAid H minus RT Kit (Thermo Fisher Scientific, Waltham, MA) using a VeritiTM 96-well thermal cycler (Applied Biosystems, Foster City, CA). We evaluated the mRNA expression of GHRH-R, GHRH, and β-actin by using the Go Taq Flexi DNA polymerase kit (Promega, Madison, WI) with primers and method described previously [4 (link), 47 (link)]. Normal rat pituitary was used as positive control. A sample that contained DNAse digested RNA and no enzyme during reverse transcription was used as negative control for RT-PCR.
In addition, total RNA from the remote area of left ventricle was isolated by the TriZol method (Invitrogen, Carlsbad, CA) as previously described [16 (link)]. We determined the mRNA expression of genes involved in ECM. Samples tested were obtained from 3–5 independent experiments.