Real-time PCR Primer Design and Validation

Cells were harvested in TRIzol (Life Technologies), extracted, digested (TURBO DNA-free Kit, Life Technologies), and cDNA synthesized (200ng–1000ng RNA, iScript cDNA Synthesis Kit, Bio-Rad) per the manufacturer’s instructions. Real-time PCR was performed on a 7900HT Fast Real-time PCR System (Applied Biosystems) with the following cycling conditions: 2 minutes at 50°C, 10 minutes at 95°C, (15 seconds at 95°C, 1 minute at 60°C) x40 cycles followed by dissociation curve (15 seconds at 95°C, 1 minute at 60°C, 15 seconds at 95°C). Human primers for PTHrP^{65 (link)}, QSOX1^{66 (link)}, PGK1^{67 (link)}, Pdcd4, and p27 coding sequence were previously published^{45 (link)}. The following primers were designed using PrimerBlast (NCBI) against the human genome (Homo sapiens) and validated by dissociation: LIFR (F-GCGTACCGACTGACTGCATTG, R-CCAGAGGGTGCTTTCCAAGAA), SOCS3 (F-GCTCCAAGAGCGAGTACCAG, R-CTGTCGCGGATCAGAAAGGT), TSP1 (F-TCCCCATCCAAAGCGTCTTC, R-ACCACGTTGTTGTCAAGGGT), TPM1 (F-TCTCAGAAGGCCAAGTCCGA, R-CAAACTCAGCCCGAGTCTCA), AMOT (F-GGCATGCCACCCCAATCT, R-TTGTAGCAAGGGCAAGGACC), TGF-β2 (F-CAGCTTGTGCTCCAGACAGT, R-GCTCAATCCGTTGTTCAGGC), P4HA1 (F-GTACATGACCCTGAGACTGGA, R-GGGGTTCATACTGTCCTCCAA), H2BK (F-CAAGGCCGTCACCAAGTACA, R-GAAGGCAATTGTGCTTCTTTTGA), IGFBP5 (F-CCCAATTGTGACCGCAAAGG, R-CGTCAACGTACTCCATGCCT), miR-190 (F-GCAGGCCTCTGTGTGATATGT, R-GGCAAGACACTGTAGGAATATGT), Selenbp1 (F-AAGTGCGAACTGGCCTTTCT, R-CCCATCCAGCAGCACAAAAC), Aldh1α1 (F-CAAGATCCAGGGCCGTACAA, R-CAGTGCAGGCCCTATCTTCC), Notch1 (F-AGCCTCAACGGGTACAAGTG, R-CACACGTAGCCACTGGTCAT), Casp3 (F-GCGGTTGTAGAAGAGTTTCGTG, R-CTCACGGCCTGGGATTTCAA), Tert (F-CTTGCGGAAGACAGTGGTGA, R-GTCCGGGCATAGCTGGAGTA), Sox2 (F-ACCAGCGCATGGACAGTTAC, R-CCGTTCATGTAGGTCTGCGA), Oct4 (F-AGAAGCTGGAGCAAAACCCG, R-ACCTTCCCAAATAGAACCCCCA), Stat3 (F-CAATACCATTGACCTGCCGAT, R-GAGCGACTCAAACTGCCCT), Socs3, (F-GCTCCAAGAGCGAGTACCAG, R-CTGTCGCGGATCAGAAAGGT), β2M (F-GAGTATGCCTGCCGTGTGAA, R-TGCGGCATCTTCAAACCTCC), and HPRT1 (F-TTGCTTTCCTTGGTCAGGCA, R-ATCCAACACTTCGTGGGGTC). Mouse HMBS was designed using PrimerBlast against the mouse genome (Mus musculus) and validated by dissociation: HMBS (F-TCATGTCCGGTAACGGCG, R-CACTCGAATCACCCTCATCTTTG).

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Johnson R.W., Finger E.C., Olcina M.M., Vilalta M., Aguilera T., Miao Y., Merkel A.R., Johnson J.R., Sterling J.A., Wu J.Y, & Giaccia A.J. (2016). Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the bone marrow. Nature cell biology, 18(10), 1078-1089.

Publication 2016

Casp3 Cdna Cells Coding sequence Genome Homo sapiens Human genome Igfbp5 Lifr Mus musculus Oct4 Primers Real time pcr Seconds Sox2 Stat3 Synthesis Tert Tgf β2 Trizol Tsp1

Corresponding Organization :

Other organizations : Stanford University, VA Tennessee Valley Healthcare System, VA MidSouth Healthcare Network

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Variable analysis

independent variables

Primer sequences used for real-time PCR

dependent variables

Expression levels of target genes (PTHrP, QSOX1, PGK1, Pdcd4, p27, LIFR, SOCS3, TSP1, TPM1, AMOT, TGF-β2, P4HA1, H2BK, IGFBP5, miR-190, Selenbp1, Aldh1α1, Notch1, Casp3, Tert, Sox2, Oct4, Stat3, Socs3, β2M, HPRT1, and mouse HMBS)

control variables

RNA input amount (200ng–1000ng)
Cycling conditions for real-time PCR (2 minutes at 50°C, 10 minutes at 95°C, (15 seconds at 95°C, 1 minute at 60°C) x40 cycles followed by dissociation curve (15 seconds at 95°C, 1 minute at 60°C, 15 seconds at 95°C))
Dissociation curve analysis to validate primer specificity

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