Fastgene scriptase 2 ready mix

The expression of Pgr, HoxA10, integrin β3 (Itgb3), and Lif mRNA in uterine tissues (one-third of one horn for 3-month-old mice and half horn for 1-month-old mice) was determined by real-time PCR. According to the manufacturer’s instructions, RNA from individual mice was isolated using an RNeasy® mini kit (Qiagen). For each sample, 1 µg of RNA was converted to cDNA using the FastGene Scriptase II Ready Mix (Nippon Genetics, Düren, Germany). The quantitative RT-PCR was performed using SYBR Green PCR Master Mix and specific primers (Table 2) in a Fast Real-time PCR (QuantStudio 3, Thermo Fisher Scientific). Relative gene expression was analyzed according to the 2^−ΔΔCt method. All samples were assayed in duplicate reactions. The Rplp0 and Gapdh genes were used as housekeeping genes to normalize the expression level, as Lin et al. advised for mouse uterus (Lin et al. 2013 (link)).
Table 2

Primers for RT-qPCR.

Gene name	GenBank number	Primer sequences (5′–3′)		Product size (bp)
		Forward	Reverse
Pgr	NM_008829.2	CTACTCGCTGTGCCTTACCATG	CTGGCTTTGACTCCTCAGTCCT	139
Hoxa10	NM_008263.4	GGCAGTTCCAAAGGCGAAAAT	GTCTGGTGCTTCGTGTAAGGC	86
Itgb3	NM_016780.2	GGCGTTGTTGTTGGAGAGTC	CTTCAGGTTACATCGGGGTCA	138
Lif	NM_008501.3	GCTGTATCGGATGGTCGCATA	CACAGACGGCAAAGCACATT	156
Gapdh	NM_001289726.2	GGTGGACCTCATGGCCTACA	CTCTCTTGATCAGTGTCCTTGCT	82
Rplp0	NM_007475.5	GGACCCGAGAAGACCTCCTT	GCACATCACTCAGAATTTCAATGG	85

For RNA extraction blood samples were collected in Tempus Blood RNA Tubes (Applied Biosystems, Foster City, CA, USA) and stored at −80 °C. RNA was extracted according to the manufacturer’s instructions. The RNA concentration was determined for each sample prior to reverse transcription using NanoDrop One (ThermoScientific, Whaltham, MA 02451, USA). Reverse transcription reactions were performed using 4 µL of the 5× FastGene^® Scriptase II ReadyMix (Nippon Genetics, Duren, Germany) and 100 ng of RNA for each reaction. Reactions were carried out in a CFX90 cycler (BioRad, Hercules, CA, USA) at the following conditions: 25 °C for 10 min, 42 °C for 60 min and 85 °C for 5 min. Real time PCR reactions were carried out in a CFX90 cycler. Each reaction consisted of 1 μL primer-probe assay mix (IDT, Coralville, IA, USA), 10 μL Luna Master Mix (Biolabs, Waltham, MA, USA) and 1 μL cDNA. The GAPDH was used as a housekeeping gene for data normalization. The sequences for the Real time PCR primers and probes for HO-1 mRNA were as follows: forward primer: 5′-GTT CCT CAT GAA CTC AGC ATT-3′, reverse primer: 5′-GAG CCA GCA CGA ACG AG-3′, probe: 56-FAM/AGC ATG CCC /ZEN/CAG GAT TTG TCA GA/3IABkFQ. Reactions were carried out in triplicate and results were analysed by the ΔΔCT method