Sybr green reaction mix

The method measuring telomere length with real-time PCR was reported previously (Peng et al., 2011 (link)). Genomic DNA was extracted on the basis of the manufacturer's instrctions (Aidlab, Beijing, China). Quantitative RT-PCR was conducted using SYBR Green Reaction Mix (TaKaRa, Dalian, China) according to the manufacturer's instructions on an ABI PRISM 7900 HT Detection System (Applied Biosystems, Carlsbad, CA, USA). The primers were as follow (Peng et al., 2011 (link)): Telomere forward primer 5′-GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3′, reverse primer 5′-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA-3′. The reference gene 36B4: forward primer 5′-CTCACTCCATCATCAATGGATACAA-3′, reverse primer 5′-CAGCCAGTGGGAAGGTGTAGTCA-3′. PCR conditions were 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 56°C for 31 s (Peng et al., 2011 (link)). The single-copy gene 36B4 was used as a control and the target gene expression was calculated using the ΔΔCt comparative method.

Total RNA was extracted from human PRLoma tissues and xenograft tumours using Trizol Reagent (Invitrogen, Carlsbad, CA, USA), according to the product manual. We reverse‐transcribed 2 μg RNA from each sample by extension of oligo primers (TaKaRa) using M‐MLV reverse transcriptase (New England Biolabs) following the manufacturer's protocol. Real‐time PCR was performed on a Bio‐Rad IQ5 cycler using a SYBR Green reaction mix (Takara). Primers are listed below. Relative expression levels were standardised to GAPDH as internal control for all real‐time PCR assays.
Hum‐ERα Forward Primer: TCTGTCTCCTGCATACACTC
Reverse Primer: GGGAATCCTCACGCTTAG
Hum‐ERβ Forward Primer: GCTTTGGTTTGGGTGATTG
Reverse Primer: CCGAGTTGATTAGAGGGTC
Hum‐GAPDH Forward Primer: TGTGGGCATCAATGGATTTGG
Reverse Primer: ACACCATGTATTCCGGGTCAAT
Rat‐ERα Forward Primer:GCTCCTAACTTGCTCTTGG
Reverse Primer:GGACTCGGTGGATGTGGT
Rat‐ERβ Forward Primer:TCTCCTTTAGCGACCCA
Reverse Primer:ACGCCGTAATGATACCC
Rat‐GAPDH Forward Primer:TTCTTGTGCAGTGCCAGCCTCGTC
Reverse Primer:TAGGAACACGGAAGGCCATGCCAG

TRIzol reagent (Invitrogen, USA) was used to extract the total RNA from cultured cells. 1-5ug of the total RNA was used for cDNA synthesis using the PrimeScript RT Reagent Kit (Takara, Japan). SYBR Green reaction mix (Takara, Japan) was used to perform the RT-qPCR assay with CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). The primers are listed in Supplementary Table S2.

Total RNA was extracted using the Trizol reagent (Life Technologies, Carlsbad, CA, USA) and first strand complementary DNA (cDNA) generated by the Reverse Transcription System (Takara) in a 20-µL reaction containing 2 µg total RNA, according to the manufacturer’s protocol. Real-time PCR was performed on a Roche LightCycler 480 using a SYBR Green reaction mix (Takara). Values are shown means and standard deviations (SD) of the results from at least three independent experiments. All primer sets are available upon request.

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then, 1 μg RNA was reverse transcribed into cDNA using the AMV retrotranscriptase system (TaKaRa, Dalian, Liaoning, China). qPCR reactions were run in triplicate on an ABI StepOne Plus System (Thermo Fisher Scientific) using SYBR Green reaction mix (TaKaRa, Dalian, Liaoning, China). The primers were designed by Primer Version 0.4.0 and listed in Supplementary Table 1. The relative expression of the target gene was calculated and normalized to the expression of the reference gene Gapdh.