T100 cycler

ERα heterozygous C57BL/6 mice (ERα^+/−) were bred and sexed hippocampi from all the pups born were cultured on an individual coverslip at P1. At the same time the toe clippings were taken for genotyping. Only the coverslips that have ERα^−/− hippocampal neurons were used for the ERα^−/− experiments. Genotypes were determined by polymerase chain reaction (PCR) of genomic DNA from finger or toe clippings: clippings were heated at 95 °C for 45 min in 50 mM NaOH and neutralized with equal volume of 1 M Tris buffer, pH 6.8. 1 µL of this DNA solution was added to 19 µL of the following: 0.25 mM of primers for the ERα gene, 1X GoTaq Buffer (Promega, Madison, WI), 0.2 mM each deoxynucleotide (Promega, Madison, WI) and 8 U Platinum Taq. PCR was performed on a BioRad T100 cycler for 30 cycles as follows: 95 °C for 3 min, denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s (ERKO PCR1) or 51 °C for 30 s (ERKO PCR2), and elongation at 72 °C for 1 min. PCR products were separated electrophoretically on an ethidium bromide-containing 2% agarose gel and visualized under UV illumination as described before [7 ].

The extracted total RNA was reverse-transcripted into cDNA using PrimeScript RT reagent Kit (TAKARA cat#RR047A) according to the manufacturer's protocol in triplicates. The resulting cDNA was pooled for next PCR amplification. Primer sequences are shown in Supplementary Table 1. The target sequences were amplified by PCR in 50μl of 1×Taq buffer containing 0.3μM of each primer, 1.5 mM magnesium chloride, 200μM dNTP mixture, 2.5 units of Taq polymerase and 1μl (10μg) of each cDNA by Ex Taq PCR kit (TAKARA cat#RR001A). The reaction was started after 5min denaturation of cDNA at 94°C (hot start). DNA amplification in a T100 cycler (Bio-Rad Laboratories, Inc) was followed by a final extension for 8min at 72°C for HJURP, ADAMTS8 and GPT2 (94°C 30s, 58.5°C 1min; 40 cycles). Although a range of annealing temperatures (from 5°C below the Tm to 5°C above the Tm) had been tried, the PCR products of other 9 mRNAs (TOP2A, GINS2, TK1, CDCA5, AGER, FHL1 CLDN18, ADH1B, and GPIHBP1) were rare compared with positive control. GAPDH expression was used as an internal control. All PCR products were visualized by 2% agarose gels electrophoresis. Water negative controls contained all components for the RT-PCR reaction without target RNA. Positive controls of RNA were extracted from A594 cells obtained from the Cancer Center of Xiamen University (Xiamen, China).

It has been reported that mouse sex can be determined at P1-3 by looking for the presence of a small dark spot between the anogenital opening which is present only in males [39 ]. To confirm this, sex genotypes were determined by PCR of DNA from tail biopsies in P0 to P1 pups [39 ]. Biopsy samples are boiled at 95 °C for 1 h in 50 mM NaOH and neutralized with equal volume of 1 M Tris buffer, pH 6.8. 20 ng/ml DNA were added to 1 pmol of primers for the Y-chromosome specific mRNASYR: and the X-chromosome mRNAMYOG [39 ] and Platinum Taq Master Mix. PCR was performed on a BioRad T100 cycler as follows: 95 °C for 3 min, denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s (PCR1) or 51 °C for 30 s (PCR2), and elongation at 72 °C for 1 min. PCR were performed for 30 cycles. PCR products were separated electrophoretically on a 2% agarose gel and visualized with ethidium bromide. In all mice that we tested the sex genotyping correlated with the visual sexing (results not shown).