Real-time reverse transcription-polymerase chain reaction (rRT-PCR) was used to quantify mammary gene expression as described by Theil et al. [21 (link)]. Briefly, total RNA was extracted from the frozen mammary biopsy after the tissue was homogenized with 350 μL RNeasy lysis buffer. The homogenate was diluted with 70% ethanol (1:1) before RNA was purified using a RNeasy mini kit (Qiagen, Albertslund, Denmark), and m-RNA was reverse-transcribed according to the manufacturer’s guide (Invitrogen, Taastrup, Denmark) using oligo-dT to synthesize cDNA. One microliter cDNA was amplified using gene-specific probes and primers with the TaqMan Universal PCR Master Mix (Applied Biosystems, Stockholm, Sweden) instrument. The signal was quantitatively detected using an ABI PRISM 7900 detection system (Applied Biosystems) to measure the labeled FAM (carboxyfluorescein) fluorophore on the 5′ end. Primer Express Version 2.0 software (Applied Biosystems) was used for primers and probe designs for all genes. Primer and probe sequences are shown in Table 2 . Gene expression of β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fatty acid synthase (FAS), delta-6 desaturase (D6D), and α-lactalbumin (α-LA) were quantified, and both β-actin and GAPDH were found to be stable housekeeping genes. The difference in cycle threshold (Ct) value between the target gene and reference genes (i.e., ΔCt-values) was used for the statistical analysis, and the relative mRNA quantity was calculated by using the formula: Relative quantity = 2−ΔΔCt.
Primer and probe sequences of target and housekeeping genes
| Genes | Accession numbers | Primer/probe | Sequence (5′ to 3′) |
|---|---|---|---|
| β-actin | AY550069 | Forward | ACCCAGATCATGTTCGAGACC |
| Reverse | TCACCGGAGTCCATCACGAT | ||
| Probe | CTGTATGCCTCTGGCCGCACCA | ||
| GAPDH | AF017079 | Forward | GTCGGAGTGAACGGATTTGG |
| Reverse | CAATGTCCACTTTGCCAGAGTTAA | ||
| Probe | CGCCTGGTCACCAGGGCTGCT | ||
| FAS | AY954688 | Forward | CGTGGGCTACAGCATGATAGG |
| Reverse | GAGGAGCAGGCCGTGTCTAT | ||
| Probe | CATCACCA | ||
| D6D | AY512561 | Forward | GACGGCCTTCATCCTTGCT |
| Reverse | ACAGAGAGATGGCCGTAATCGT | ||
| Probe | CCTCTCAGGCCCAGGCTGGGTG | ||
| α-LA | M80520 | Forward | ACAATGGCAGCACAGAATATGG |
| Reverse | TCAGTAAGGTCATCATCCAGGAATT | ||
| Probe | CTCTTCCAGATCAATAAT |
GAPDH Glyceraldehyde-3-phosphate dehydrogenase, FAS Fatty acid synthase, D6D Delta-6 desaturase, α-LA α-lactalbumin
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