Real-time PCR

Tumor samples, right lung lobes, brains, and intact right femora were homogenized in 1 ml TRIzol (Life Technologies), spun down to clear the lysate, phenol–chloroform extracted, DNase digested (TURBO DNA-free Kit, Life Technologies), and cDNA synthesized (1 μg RNA, iScript cDNA Synthesis Kit, Bio-Rad) per the manufacturer’s instructions. Real-time PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) on a QuantStudio 5 (Thermo Fisher) with the following conditions: 2 min at 50 °C, 10 min at 95 °C, (15 s at 95 °C, 1 min at 60 °C) × 40 cycles followed by dissociation curve (15 s at 95 °C, 1 min at 60 °C, 15 s at 95 °C). For each biological replicate, three technical replicates were performed for each gene analyzed. Non-template controls were included as a negative control for each gene analyzed. Analysis was performed by normalizing the expression of the target gene to the average Hmbs or B2m expression within the same sample to determine ΔCt. The ΔCt was transformed (2^−ΔCt) and the average of the three technical replicates was calculated. The average 2^−ΔCt for each mouse is presented as target gene “(Pymt, Hif1a, Hif2a, etc.): (B2m or Hmbs) mRNA” or “Relative mRNA abundance” in the figures. Mouse primers for PyMT⁹⁵ (Pymt—F: CTGCTACTGCACCCAGACAA, R: GCAGGTAAGAGGCATTCTGC) and hydroxymethylbilane synthase⁹⁶ (Hmbs—F: TCATGTCCGGTAACGGCG, R: CACTCGAATCACCCTCATCTTTG) were previously published. Primer sequences for parathyroid hormone-related protein (Pthlh—F: ACATTGCTATGGGAGCCAC, R: TAGGAATCAGCGCCTCTAAC) were kindly provided by Dr. Natalie Sims and Dr. T. John Martin at St. Vincent’s Institute of Medical Research. Primers for beta-2-microglobulin, VEGFA, Hif1α (exon 2), Hif2α (exon 2), and Vhl (exon 1) were designed using PrimerBlast (NCBI) against the mouse genome (Mus musculus) and validated by dissociation: B2m (F: TTCACCCCCACTGAGACTGAT, R: GTCTTGGGCTCGGCCATA), Vegfa (F: GGAGATCCTTCGAGGAGCACTT, R: GGCGATTTAGCAGCAGATATAAGAA), Hif1α (F: TCGGCGAAGCAAAGAGTCTG, R: GCTCACATTGTGGGGAAGTG), Hif2α (F: TGAGGAAGGAGAAATCCCGTG, R: GGGCAACTCATGAGCCAACT), Vhl (F: GACCCGTTCCAATAATGCCC, R: CGTCGAAGTTGAGCCACAAA). Additional primer sequences were obtained from the Massachusetts General Hospital PrimerBank and specificity was confirmed by nucleotide BLAST (NCBI) and validated by dissociation: Cdh1 (F: CAGGTCTCCTCATGGCTTTGC, R: CTTCCGAAAAGAAGGCTGTCC), Cdh2 (F: AGCGCAGTCTTACCGAAGG, R: TCGCTGCTTTCATACTGAACTTT), Egfr (F: ATGAAAACACCTATGCCTTAGCC, R: TAAGTTCCGCATGGGCAGTTC), Notch1 (F: GATGGCCTCAATGGGTACAAG, R: TCGTTGTTGTTGATGTCACAGT), Snai1 (F: CACACGCTGCCTTGTGTCT, R: GGTCAGCAAAAGCACGGTT), Snai2 (F: CATCCTTGGGGCGTGTAAGTC, R: GCCCAGAGAACGTAGAATAGGTC), Snai3 (F: GGTCCCCAACTACGGGAAAC, R: CTGTAGGGGGTCACTGGGATT), Twist1 (F: GGACAAGCTGAGCAAGATTCA, R: CGGAGAAGGCGTAGCTGAG), Zeb1 (F: ACTGCAAGAAACGGTTTTCCC, R: GGCGAGGAACACTGAGATGT), Zeb2 (F: ATTGCACATCAGACTTTGAGGAA, R: ATAATGGCCGTGTCGCTTCG), Atg7 (F: TCTGGGAAGCCATAAAGTCAGG, R: GCGAAGGTCAGGAGCAGAA), Becn1 (F: ATGGAGGGGTCTAAGGCGTC, R: TCCTCTCCTGAGTTAGCCTCT), Map1lc3a (F: GACCGCTGTAAGGAGGTGC, R: CTTGACCAACTCGCTCATGTTA), Map1lc3b (F: TTATAGAGCGATACAAGGGGGAG, R: CGCCGTCTGATTATCTTGATGAG), Sqstm1 (F: GAACTCGCTATAAGTGCAGTGT, R: AGAGAAGCTATCAGAGAGGTGG), Ulk1 (F: AAGTTCGAGTTCTCTCGCAAG, R: CGATGTTTTCGTGCTTTAGTTCC), Pgk1 (F: TGGTGGGTGTGAATCTGCC, R: ACTTTAGCGCCTCCCAAGATA), Glut1 (F: CAGTTCGGCTATAACACTGGTG, R: GCCCCCGACAGAGAAGATG), Tek (F: CTGGAGGTTACTCAAGATGTGAC, R: TCCGTATCCTTATAGCCTGTCC), Pdgfb (F: CATCCGCTCCTTTGATGATCTT, R: GTGCTCGGGTCATGTTCAAGT), Cxcr4 (F: GAGGCCAAGGAAACTGCTG, R: GCGGTCACAGATGTACCTGTC). The expression of a panel of metastasis-associated genes was quantified using the tumor metastasis (SAB Target List, M384) qPCR array plate (Bio-Rad). Each gene in the array was run in duplicate. Three representative Hif1α^f/f and Hif1α^−/− tumor homogenate RNA samples were selected based on their Hif1α expression. Analysis was performed by normalizing the expression of the target gene to the average B2m expression within the same sample to determine ΔCt. The ΔCt was transformed (2^−ΔCt) and the average of the two technical replicates was calculated. ΔCt values for each Hif1α^−/− sample was then normalized against the average of the three Hif1α^f/f samples to determine enrichment.