H & E sections from all available FFPE tissue specimens were evaluated histologically by a certified pathologist who recorded percentage of tumor cell content in each one. Prior to RNA isolation, macrodissection of tumor areas was performed in most of the FFPE sections with <50% tumor cell content. The tumor cell content was >30% in practically all (97%) of the samples and >50% in the majority (76%) of the samples. More than one FFPE section was used for RNA extraction when the tumor surface of a given sample was less than 0.25 cm2, in an effort to minimize the rate of technical failures in the RNA extraction.
Sufficient RNA was isolated from 308 FFPE specimens followed by qRT-PCR, as previously described [31 (link)]. From each FFPE section or macrodissected tissue fragment (10 μm thick), RNA was isolated using a standardized fully automated isolation method for total RNA from FFPE tissue, based on silica-coated magnetic beads (VERSANT Tissue Preparation Reagents, Siemens Healthcare Diagnostics, Tarrytown, NY, USA) in combination with a liquid handling robot, as previously described in detail [22 (link)]. The method involves extraction-integrated deparaffinization and DNase I digestion steps. DNA-free total RNA was eluted with 100 μL elution buffer and stored at -80°C.
One-step qRT-PCR was applied for the relative quantification of VEGF-A, VEGF-B, VEGF-C, VEGFR1, VEGFR2 and VEGFR3 mRNA expression, by using gene-specific TaqMan® based assays. Forty cycles of nucleic acid amplification were applied and the cycle threshold (CT) values of the target genes were identified. CT values were normalized by subtracting the CT value of the housekeeping gene RPL37A (ribosomal protein L37a) from the CT value of the target genes (ΔCT). RNA results were then reported as 40-ΔCT values, which correlate proportionally with the mRNA expression level of the target genes. For assessment of DNA contamination, a qPCR analysis specific for the PAEP gene (progestagen-associated endometrial protein) was performed, without the preceding reverse-transcription step. Samples were considered to be substantially free of DNA when CT values above 38 were detected. In the case of DNA contamination, samples were manually re-digested with DNase I. The quantity of RNA following isolation (yield) was checked by measuring RPL37A expression as a surrogate marker for amplifiable mRNA. Samples with average RPL37A CT values <32 were considered to have sufficient RNA and were eligible for analysis. Only 3 of the 311 extracted samples (1%) had an average RPL37A CT value of ≥32 and were, therefore, excluded from further analysis, resulting in successful RNA extraction from 99% of the samples.
Expression of the target genes, as well as the reference gene RPL37A, was assessed in triplicate by qRT-PCR using the SuperScript III PLATINUM One-Step Quantitative RT-PCR System with ROX (Invitrogen, Karlsruhe, Germany) in an ABI PRISM 7900HT (Applied Biosystems, Darmstadt, Germany) [21 (link)]. The lengths of the amplicons detected by the VEGF-A, VEGF-B, VEGF-C, VEGFR1, VEGFR2, VEGFR3 and RPL37A assays were 80 bp, 81 bp, 77 bp, 85 bp, 68 bp, 70 bp and 65 bp, respectively, with PCR efficiencies [E = 1(10-slope)] of 85.5, 110.3, 88.2, 95.7, 94.3, 84.7 and 86.0%, respectively. A commercially available human reference RNA (Stratagene qPCR Human Reference Total RNA, Agilent Technologies, Waldbronn, Germany) was used as positive control. No-template controls were assessed in parallel to exclude contamination.
The Primer/Probe (FAM/TAMRA-labeled) sets used for amplification of the target and reference genes were the following (5' -> 3'):
VEGF-A Probe CACCATGCAGATTATGCGGATCAAACCT
Forward Primer GCCCACTGAGGAGTCCAACA
Reverse Primer TCCTATGTGCTGGCCTTGGT
VEGF-B Probe CACATCTATCCATGACACCACTTTCCTCTGG
Forward Primer TGGCAGGTAGCGCGAGTAT
Reverse Primer CCCTGTCTCCCAGCCTGAT
VEGF-C Probe TTGAGTCATCTCCAGCATCCGAGGAAA
Forward Primer CCACAGATGTCATGGAATCCAT
Reverse Primer TGCCTGGCTCAGGAAGATTT
VEGFR1 Probe TGCTGTCGCCCTGGTAGTCATCAAACA
Forward Primer CATGGGAGAGGCCAACAGA
Reverse Primer AACCTTTGAAGAACTTTTACCGAATG
VEGFR2 Probe TCTTGGCATCGCGAAAGTGTATCCACA
Forward Primer TTCCAAGTGGCTAAGGGCAT
Reverse Primer CGTGCCGCCAGGTCC
VEGFR3 Probe TGCCTGCTTCCCTGGGTAGTCCC
Forward Primer GCACCCACTTACCCCGC
Reverse Primer GAGTTTAACTCAGGTGTCACCTTTGA
RPL37A Probe TGGCTGGCGGTGCCTGGA
Forward Primer TGTGGTTCCTGCATGAAGACA
Reverse Primer GTGACAGCGGAAGTGGTATTGTAC
Sufficient RNA was isolated from 308 FFPE specimens followed by qRT-PCR, as previously described [31 (link)]. From each FFPE section or macrodissected tissue fragment (10 μm thick), RNA was isolated using a standardized fully automated isolation method for total RNA from FFPE tissue, based on silica-coated magnetic beads (VERSANT Tissue Preparation Reagents, Siemens Healthcare Diagnostics, Tarrytown, NY, USA) in combination with a liquid handling robot, as previously described in detail [22 (link)]. The method involves extraction-integrated deparaffinization and DNase I digestion steps. DNA-free total RNA was eluted with 100 μL elution buffer and stored at -80°C.
One-step qRT-PCR was applied for the relative quantification of VEGF-A, VEGF-B, VEGF-C, VEGFR1, VEGFR2 and VEGFR3 mRNA expression, by using gene-specific TaqMan® based assays. Forty cycles of nucleic acid amplification were applied and the cycle threshold (CT) values of the target genes were identified. CT values were normalized by subtracting the CT value of the housekeeping gene RPL37A (ribosomal protein L37a) from the CT value of the target genes (ΔCT). RNA results were then reported as 40-ΔCT values, which correlate proportionally with the mRNA expression level of the target genes. For assessment of DNA contamination, a qPCR analysis specific for the PAEP gene (progestagen-associated endometrial protein) was performed, without the preceding reverse-transcription step. Samples were considered to be substantially free of DNA when CT values above 38 were detected. In the case of DNA contamination, samples were manually re-digested with DNase I. The quantity of RNA following isolation (yield) was checked by measuring RPL37A expression as a surrogate marker for amplifiable mRNA. Samples with average RPL37A CT values <32 were considered to have sufficient RNA and were eligible for analysis. Only 3 of the 311 extracted samples (1%) had an average RPL37A CT value of ≥32 and were, therefore, excluded from further analysis, resulting in successful RNA extraction from 99% of the samples.
Expression of the target genes, as well as the reference gene RPL37A, was assessed in triplicate by qRT-PCR using the SuperScript III PLATINUM One-Step Quantitative RT-PCR System with ROX (Invitrogen, Karlsruhe, Germany) in an ABI PRISM 7900HT (Applied Biosystems, Darmstadt, Germany) [21 (link)]. The lengths of the amplicons detected by the VEGF-A, VEGF-B, VEGF-C, VEGFR1, VEGFR2, VEGFR3 and RPL37A assays were 80 bp, 81 bp, 77 bp, 85 bp, 68 bp, 70 bp and 65 bp, respectively, with PCR efficiencies [E = 1(10-slope)] of 85.5, 110.3, 88.2, 95.7, 94.3, 84.7 and 86.0%, respectively. A commercially available human reference RNA (Stratagene qPCR Human Reference Total RNA, Agilent Technologies, Waldbronn, Germany) was used as positive control. No-template controls were assessed in parallel to exclude contamination.
The Primer/Probe (FAM/TAMRA-labeled) sets used for amplification of the target and reference genes were the following (5' -> 3'):
VEGF-A Probe CACCATGCAGATTATGCGGATCAAACCT
Forward Primer GCCCACTGAGGAGTCCAACA
Reverse Primer TCCTATGTGCTGGCCTTGGT
VEGF-B Probe CACATCTATCCATGACACCACTTTCCTCTGG
Forward Primer TGGCAGGTAGCGCGAGTAT
Reverse Primer CCCTGTCTCCCAGCCTGAT
VEGF-C Probe TTGAGTCATCTCCAGCATCCGAGGAAA
Forward Primer CCACAGATGTCATGGAATCCAT
Reverse Primer TGCCTGGCTCAGGAAGATTT
VEGFR1 Probe TGCTGTCGCCCTGGTAGTCATCAAACA
Forward Primer CATGGGAGAGGCCAACAGA
Reverse Primer AACCTTTGAAGAACTTTTACCGAATG
VEGFR2 Probe TCTTGGCATCGCGAAAGTGTATCCACA
Forward Primer TTCCAAGTGGCTAAGGGCAT
Reverse Primer CGTGCCGCCAGGTCC
VEGFR3 Probe TGCCTGCTTCCCTGGGTAGTCCC
Forward Primer GCACCCACTTACCCCGC
Reverse Primer GAGTTTAACTCAGGTGTCACCTTTGA
RPL37A Probe TGGCTGGCGGTGCCTGGA
Forward Primer TGTGGTTCCTGCATGAAGACA
Reverse Primer GTGACAGCGGAAGTGGTATTGTAC
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