A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE Samples

Abstract

BackgroundReverse transcription quantitative real-time PCR (RT-qPCR) tests support personalized cancer treatment through more clinically meaningful diagnosis. However, samples obtained through standard clinical pathology procedures are formalin-fixed, paraffin-embedded (FFPE) and yield small samples with low integrity RNA containing PCR interfering substances. RT-qPCR tests able to assess FFPE samples with quality control and inter-laboratory reproducibility are needed.MethodsWe developed an RT-qPCR method by which 1) each gene was measured relative to a known number of its respective competitive internal standard molecules to control for interfering substances, 2) two-color fluorometric hydrolysis probes enabled analysis on a real-time platform, 3) external standards controlled for variation in probe fluorescence intensity, and 4) pre-amplification maximized signal from FFPE RNA samples. Reagents were developed for four genes comprised by a previously reported lung cancer diagnostic test (LCDT) then subjected to analytical validation using synthetic native templates as test articles to assess linearity, signal-to-analyte response, lower detection threshold, imprecision and accuracy. Fitness of this method and these reagents for clinical testing was assessed in FFPE normal (N = 10) and malignant (N = 10) lung samples.ResultsReagents for each of four genes, MYC, E2F1, CDKN1A and ACTB comprised by the LCDT had acceptable linearity (R2>0.99), signal-to-analyte response (slope 1.0±0.05), lower detection threshold (<10 molecules) and imprecision (CV <20%). Poisson analysis confirmed accuracy of internal standard concentrations. Internal standards controlled for experimentally introduced interference, prevented false-negatives and enabled pre-amplification to increase signal without altering measured values. In the fitness for purpose testing of this two-color fluorometric LCDT using surgical FFPE samples, the diagnostic accuracy was 93% which was similar to that previously reported for analysis of fresh samples.ConclusionsThis quality-controlled two-color fluorometric RT-qPCR approach will facilitate the development of reliable, robust RT-qPCR-based molecular diagnostic tests in FFPE clinical samples.

Operator Dependent Choice of Prostate Cancer Biopsy Has Limited Impact on a Gene Signature Analysis for the Highly Expressed Genes IGFBP3 and F3 in Prostate Cancer Epithelial Cells

“…An advantage of FFPE samples is that they can be easily archived and that many cohorts have long time follow-up clinical data available, which greatly facilitates clinical studies. Even though the extracted RNA from FFPE samples may be of relatively low quality, multiple recent studies have shown promising results when utilizing degraded RNA extracted from archival FFPE samples for quantifying gene expression levels by optimized qRT-PCR methods [5] – [7] . One example is the Prostatype qRT-PCR kit, which is developed and optimized for measuring the gene expression levels of a gene signature of IGFBP3, F3 and VGLL3 particularly in FFPE samples.…”

A high-throughput method to detect RNA profiling by integration of RT-MLPA with next generation sequencing technology

“…Due to the probe length limitation, RT-MLPA can only detect up to 45 targets [ 12 ] or 90-100 host genes in a single reaction using dual-color assay (dcRT-MLPA) [ 14 ]. To improve the throughput and simplify the operating process, we applied Next-Generation Sequencing (NGS) technology to replace the capillary electrophoresis in RT-MLPA method.…”

“…After PCR with P5/P7 adaptor primers RT-MLPSeq Libraries from FFPE RNA were successfully prepared ( Supplementary Figure 2 ) and sequenced. Meanwhile we applied RT-qPCR methods to measure RNA expression as previous reported [ 14 ] and compared with results derived from RT-MLPSeq for the same sample. The mRNA expression levels of 16 cancer-associated genes (Ki67, STK15, Survivin, CCNB1, MYBL2, GRB7, HER2, MMP11, CTSL2, ER, PGR, BCL2, SCUBE2, GSTM1, CD68, BAG1) detected by both methods were normalized to 5 reference genes (ACTB, GAPDH, RPLPO, GUSB, TFRC).…”

“…Currently the most commonly used RNA quantification methods in FFPE samples were RT-qPCR [ 4 , 5 , 14 ]. To our best knowledge, the length of RNA from FFPE samples can be as small as 80 bps [ 6 ], which could be reverse-transcribed to cDNA larger than 60 bps.…”

“…The other feature of RT-MLPSeq was high-throughput for target RNA. Unlike traditional RT-MLPA [ 12 ] or dual-color RT-MLPA [ 14 ] which distinguish different targets with unique length of ligated probe pairs, NGS can analyze the specific nucleotide sequences in LHS and RHS and thus have no limit on the number of probes in one reaction.…”