Perfecta sybr green supermix for iq

Total RNA was isolated from tissue samples using TRI Reagent (Sigma-Aldrich, St. Louis, MO), and then converted to cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermofisher). Quantitative real-time PCR was conducted using PerfeCTa SYBR Green Supermix for iQ (Quanta Biosciences, Beverly, MA) on a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Primers used for real-time PCR are listed in Table S1. Ribosomal protein, large, P0 (Rplp0) was used to normalize gene expression.

Embryos and decidua-free placentas (E12.5) were isolated in cold PBS and flash frozen in liquid nitrogen. RNA was isolated using Trizol (Invitrogen) and reverse transcribed using SuperScript II (Quanta Biosciences). Gene expression was measured quantitatively using PerfeCTa SYBR Green SuperMix for iQ (Quanta Biosciences). Primer sets are listed in the table shown below. Differences among target expression were quantified using the ΔΔCT method and normalized to ß-actin and 18S rRNA.
For analysis of miR-126 expression, 10ng of RNA was reverse transcribed using TaqMan MicroRNA Reverse Transcription Kit (Life Technologies #4366596) with RT primers specific for miR-126 (002228) and control snoRNA234 (0001234). Real Time PCR was performed using TaqMan Universal PCR Master Mix (Life Technologies #4324018) and TaqMan probes for miR-126 and control snoRNA234.

Trizol reagent (Invitrogen; Carlsbad, CA) was used to extract total RNA
from GMs according to manufacturer’s instructions. Phenol/chloroform
extraction and ethanol precipitation sequentially followed the extraction.
Contaminating DNA was degraded by two 10 min treatments with DNase I (ProMega;
Madison, WI) and the DNase was subsequently heat inactivated. A
GoScript™ Reverse Transcriptase Kit (ProMega; Madison, WI) was used to
reverse transcribe cDNA using an Eppendorf Mastercycler Personal (Hauppauge,
NY). PerfeCTa SYBR Green Supermix for iQ (Quanta Biosciences, Inc.;
Gaithersberg, MD) was added to the PCR reaction (per manufacturer’s
instructions) and primers (see Table 1) for the gene of interest were added to
the samples (in triplicate) while primers for an appropriate
“housekeeping” gene (the 18S ribosomal subunit) were added to
identical samples (in triplicate). A BioRad MyiQ single Color Real time PCR
Detection System (BioRad; Hercules, CA) was used to detect SYBR Green
fluorescence as a measure of amplicon. Sample CT values were normalized to
(subtracted from) the CT values of the 18S “housekeeping” gene
and the number 2 was raised to a power equal to the difference between the
sample CT values of the 18S subunit and the gene of interest.^{22 (link),23 (link),25 (link)}