Validation of Putative Reference Genes for Normalization of Q-RT-PCR Data From Paraffin-embedded Lymphoid Tissue

Abstract

Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. This study aimed to determine the most appropriate reference genes for normalizing gene expressions in lymphatic tissue, represented by non-neoplastic lymph nodes and diffuse large B-cell lymphomas, by using 2 statistical software applications, geNorm and NormFinder. In addition, we wanted to validate the usefulness of paraffin-embedded samples for Q-RT-PCR studies by investigating gene expressions of relevant target genes in paired frozen and paraffin-embedded samples. Moreover, we studied the impact of amplicon sizes on the efficiency of Q-RT-PCR in paraffin-embedded tissues. Six putative reference genes were tested for stability of expression in 21 pairs of snap-frozen and formalin-fixed, paraffin-embedded lymph nodes and lymphomas. The genes were ranked according to their suitability as reference genes. According to both statistical approaches, beta-glucoronidase was the single most appropriate reference gene in both snap-frozen and paraffin-embedded samples. TATA box-binding protein gene and Abelson murine leukemia viral oncogene homolog 1 gene were also highly ranked by both programs. In addition, we measured the relative expressions of 7 target genes by Q-RT-PCR, using PCR primer-probes with amplicon sizes up to 105 bases. The correlation coefficient for expression measured in matched frozen and paraffin-embedded samples was 0.93 (P<0.01) after normalization with the appropriate reference genes. Thus, we show that formalin-fixed, paraffin-embedded lymphoid samples are suitable for Q-RT-PCR when using thoroughly validated reference genes.

Gene expression profiles in granuloma tissue reveal novel diagnostic markers in sarcoidosis

“…This approach has demonstrated clinical utility (Clark-Langone et al, 2010). Our study utilized several additional measures to validate the technique, including primers designed to span introns, primers that are non-homologous with other human cDNA sequences, and inclusion of negative and positive controls (cDNA vectors with verified DNA sequence of each gene) for each reaction (April et al, 2009; Green et al, 2009; Sanchez-Navarro et al, 2010). Furthermore, the generated PCR product was confirmed with examination of melting peaks and by agarose gel electrophoresis (Christophi et al, 2012b).…”

Accuracy of Molecular Data Generated with FFPE Biospecimens: Lessons from the Literature

“…Other studies report equivalent performance among differentially preserved biospecimens (1,28,29). Of the studies that reported decreased success in FFPE biospecimens, differences were attributed to RNA degradation, leading to alterations in transcript abundance and length (2,3,23,24). Importantly, such alterations are not global, as transcript-specific effects have been noted (30).…”

“…Analytical parameters such as amplicon size and priming method can be manipulated to improve assay success. While RT-PCR success with FFPE biospecimens is influenced by amplicon size (2,23,24,28), reliable qRT-PCR data has been obtained with both FFPE and frozen biospecimens for amplicons between 54 and 105 bp in length (2). One study reported that low percent present rates for FFPE biospecimens increased when samples were primed with both oligo dT and random hexamer primers and analyzed by Exon arrays (25), while another study reported an increase following transcript repair (24).…”

“…Despite differences in raw cycle threshold (Ct) values of 3-10 cycles between FFPE and frozen biospecimens, a strong correlation (r=0.93) has been achieved when levels are normalized to one or more transcript(s) (2). Notably, transcript- and tumor-specific effects necessitate the evaluation of individual transcripts and tumor types.…”

“…However, when RNA amplification was preceded by a complementary-template reverse transcription repair step, in FFPE biospecimens, it resulted in longer transcripts than those obtained with untreated FFPE controls (maximum length: 750 bp versus <200 bp), although a number of tissue-specific transcripts were lost (24). Differences in relative expression between FFPE and frozen tissue were also influenced by the number of transcripts used for normalization of real time qRT-PCR (2), and while the strongest correlations were reported with 2 normalization transcripts, transcript choice and the number used are not universal, and verification by comparison with frozen biospecimens is still required (2). …”