Separate Quality-Control Measures Are Necessary for Estimation of RNA and Methylated DNA from Formalin-Fixed, Paraffin-Embedded Specimens by Quantitative PCR

Abstract

Estimations of RNA abundance and DNA methylation by quantitative PCR (qPCR) from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are not yet routine in clinical laboratory practice. Excluding specimens with poorly preserved nucleic acids is an important quality-control step for avoiding unreliable results. Because the assays for RNA abundance and DNA methylation have different critical limiting factors, we examined the extent of overlap of excluded specimens for RNA abundance versus methylated DNA. The transcript abundance of three reference genes and of the test gene, estrogen receptor 1 (ESR1), was estimated by SYBR Green qPCR in 250 breast cancer specimens. The estrogen receptor (ER) protein was identified by IHC, and concordance between ESR1 and ER was estimated by Cohen's κ. TaqMan PCR for the ALU-C4 sequence was performed with bisulfite-treated DNA to determine usability in the MethyLight assay. Excluding specimens with mean reference gene CT values exceeding the group mean by >1 SD led to significant improvement of the concordance of ESR1 and ER. Specimens with usable DNA after bisulfite treatment likewise had ALU-C4 CT values of less than the group mean + 1 SD. Samples with low-quality RNA and DNA were partly nonoverlapping. RNA and DNA extracted from the same FFPE block need separate exclusion criteria for qPCR assays of transcript abundance and methylated DNA.

HR+HER2− breast cancers with growth factor receptor–mediated EMT have a poor prognosis and lapatinib downregulates EMT in MCF-7 cells

“…23 For cell line experiments, trypsinized (0.05% trypsin/EDTA) cells were pelleted and subjected to digestion at 55°C for 1 h with Tris-EDTA (TE) buffer containing 1% sodium dodecyl sulfate (SDS) and Proteinase K (Qiagen), followed by the same protocol as used for tumor samples.…”

Dissecting the Biological Heterogeneity within Hormone Receptor Positive HER2 Negative Breast Cancer by Gene Expression Markers Identifies Indolent Tumors within Late Stage Disease

“…Tumors from 140 patients (including 4 bilateral) were used for the final analysis and the process of progressive exclusion of samples from the analysis is shown in Supplementary Figure S1. Only blocks with greater than 50% cancer epithelial cells which passed our molecular QC [24] were used for the analysis. Information on the hormone receptor status like ER, PR and HER2 was obtained from hospital records.…”

“…Total RNA was extracted from two 20 μm sections taken from each patient's tumor block as described before [24]. Briefly, sections were deparaffinized by heat, and then subjected to overnight digestion using proteinase K (Qiagen #19133).…”

“…500 ng of total RNA was then reverse transcribed using the ABI high capacity cDNA archive kit (ABI # 4322171) as per the manufacturer's protocol. The methods used for nucleic acid extraction, qRT-PCR, selection of housekeeping genes (HKG), and quality control criteria for inclusion of samples in this analysis have been described in detail in a previous publication from our laboratory [24].…”

“…Pre incubation and initial denaturation of the template cDNA were performed at 95 °C for 10 minutes, followed by amplification for 45 cycles at 95 °C for 15 seconds and 60 °C for 1 minute. Quality control criteria of cycle of threshold (Ct) values less than 32.25 were used for the selection of samples as detailed earlier [24]. Ct values for the test genes were in turn normalized relative to the mean Ct value of the three reference genes for each sample as ∆Ct.…”