Table 1 RT-qPCR gene, primer, target and amplicon specifications for reference genes (PPIB, RPL22) and target genes

From: The role of mechanotransduction versus hypoxia during simulated orthodontic compressive strain—an in vitro study of human periodontal ligament fibroblasts

Gene symbol

Gene name (Homo sapiens)

Accession numbera (NCBI GenBank)

Chromosomal locationa (length)

5′-forward primer-3′ (length/ Tm/ %GC/max. ∆G Hairpin &Self-Dimer/ Self-Comp./ Self-3′-Comp.)

5′-reverse primer-3′ (length/Tm /%GC/max. ∆G Hairpin &Self-Dimer/Self-Comp./ Self-3′-Comp.)

Primer locationb (max. ∆G Cross-Dimer)

Amplicon (length, %GC, Tm, SSAT)

Amplicon location (bp of Start/Stop)

Intron spanningb (length)

In silico qPCR specificityc

Variants targeted (transcript/splice)b

PPIB

Peptidylprolyl isomerase B

NM_000942.4

15q22.31 (1 045 bp)

TTCCATCGTGTAATCAAGGACTTC (24 bp/ 61.3 °C/ 41.7%/–1.3/4/ 2)

GCTCACCGTAGATGCTCTTTC (21 bp/ 61.2 °C/ 52.4%/–0.7/4 /0)

Exon ¾ (–2.1)

88 bp, 53.4%, 86.1 °C, no SSAT

446/533

Yes (3 194 bp)

Yes (BLAST/UCSC)

Yes

RPL22

Ribosomal protein L22

NM_000983.3

1p36.31 (2 099 bp)

TGATTGCACCCACCCTGTAG (20 bp/ 62.2 °C/ 55.0%/–3.4/4/2)

GGTTCCCAGCTTTTCCGTTC (20 bp/61.8 °C/55.0%/–3.0/ 4/ 0)

Exon 2/3 (–1.5)

98 bp, 44.9%, 83.8 °C, no SSAT

115/212

Yes (4 597 bp)

Yes (BLAST/UCSC)

Yes

ALPL

Alkaline phosphatase, liver/bone/kidney

NM_000478.5

1p36.12 (2 613 bp)

ACAAGCACTCCCACTTCATCTG (22 bp/ 60.3 °C/ 50.0%/–0.5/3/ 2)

GGTCCGTCACGTTGTTCCTG (20 bp/61.4 °C/60.0%/–3.3/ 5/ 1)

Exon 7–8/9 (–2.1)

132 bp, 56.1%, 89.5 °C, no SSAT

1 045/1 176

Yes (3 290 bp)

Yes (BLAST/UCSC)

Yes

COL1A2

Collagen type I alpha-2 chain

NM_000089.3

7q21.3 (5 411 bp)

AGAAACACGTCTGGCTAGGAG (21 bp/ 59.8 °C/52.4%/–3.3/4/2)

GCATGAAGGCAAGTTGGGTAG (21 bp/59.8 °C/52.4%/–2.3/5 /0)

Exon 50/51 (–0.7)

105 bp, 44.8%, 83.3 °C no SSAT

4 139/4 243

Yes (7 10 bp)

Yes (BLAST/UCSC)

Yes

COX-2 (PTGS2)

Prostaglandin-endoperoxide synthase 2

NM_000963.3

1q31.1 (4 507 bp)

GAGCAGGCAGATGAAATACCAGTC (24 bp/62.7 °C/ 50.0%/ 0.0/2/ 2)

TGTCACCATAGAGTGCTTCCAAC (23 bp/60.6 °C/47.8%/–1.3/4 /0)

Exon 8/9 (–3.2)

131 bp, 42.0%, 82.9 °C, no SSAT

1 457/1 587

Yes (4 86 bp)

Yes (BLAST/UCSC)

Yes

VEGFA

Vascular endothelial growth factor A

NM_001171623.1

6p21.1 (3 677 bp)

TGCAGACCAAAGAAAGATAGAGC (23 bp/ 58.9 °C/ 43.5%/–3.4/4/ 2)

ACGCTCCAGGACTTATACCG (20 bp/59.4 °C/55.0%/–1.3/ 5/ 2)

Exon 5–6/7 (–3.3)

107 bp, 43.9%, 83.7 °C, no SSAT

1 426/1 532

No

Yes (BLAST/UCSC)

Yes

TNFRSF11B (OPG)

Tumour necrosis factor receptor superfamily member 11b (osteoprotegerin)

NM_002546.3

8q24.12 (2 354 bp)

TGTCTTTGGTCTCCTGCTAACTC (23 bp/ 60.6 °C/47.8%/0.0/2/ 0)

CCTGAAGAATGCCTCCTCACAC (22 bp/62.1 °C/54.6%/–0.9/4 /0)

Exon 3/4 (–1.8)

124 bp, 42.7%, 83.1 °C no SSAT

824/947

Yes (2 019 bp)

Yes (BLAST/UCSC)

Yes

TNFSF11 (RANKL)

Tumour necrosis factor superfamily member 11 (receptor activator of nuclear factor kappa B)

NM_003701.3

13q14.11 (2 216 bp)

ATACCCTGATGAAAGGAGGA (20 bp/54.9 °C/45.0%/–1.3/3/ 0)

GGGGCTCAATCTATATCTCG (20 bp/54.6 °C/50.0%/–0.5/ 4/ 2)

Exon 8 (–2.0)

202 bp, 43.6%, 82.5 °C, no SSAT

907/1 108

No

Yes (BLAST/UCSC)

Yes

  1. Primer secondary structures (hairpin, dimers) at annealing temperature were determined by BeaconDesigner (Premier Biosoft International, http://www.premierbiosoft.com/qOligo/Oligo.jsp?PID = 1). Primers for TNFSF11 were previously published by Nettelhoff et al.22
  2. Tm, melting temperature of primer/specific qPCR product (amplicon); %GC, guanine/cytosine content; bp, base pairs; Comp., complementarity; SSAT, secondary structure at annealing temperature (determined with UNAFold; https://eu.idtdna.com/UNAFold?)
  3. aPrimer design based on this sequence. The database source was the NCBI Nucleotide database (http://www.ncbi.nlm.nih.gov/nuccore)
  4. bDetermined with PrimerCheck (http://projects.insilico.us/SpliceCenter/PrimerCheck; SpliceCenter)
  5. cDetermined in silico by NCBI PrimerBLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast) and UCSC In-Silico PCR (http://genome.ucsc.edu/cgi-bin/hgPcr)
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