Comparison of Two Methods of RNA Extraction from Formalin-Fixed Paraffin-Embedded Tissue Specimens

Gouveia, Gisele Rodrigues; Ferreira, Suzete Cleusa; Ferreira, Jerenice Esdras; Siqueira, Sheila Aparecida Coelho; Pereira, Juliana

doi:10.1155/2014/151724

Cited by 23 publications

(19 citation statements)

References 12 publications

(23 reference statements)

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“…Already in the 90s, Ben-Ezra et al showed that after six hours of tissue fixation by the Bouin solution, the PCR gave no result [16]. Later, this conclusion has been questioned by a team in Brazil [17] who showed with two different protocols that it was possible to get between 33.3% and 80% positivity to PCR despite the use of the Bouin solution. This same team demonstrated that formalin-fixed tissues gave PCR positivity rates between 87.5% and 100%; and the use of formaldehyde as a fixative gave between 17.6% and 73.7% of positivity.…”

Section: Discussionmentioning

confidence: 99%

Distribution of High-Risk Human Papillomavirus Genotypes in Precancerous Cervical Lesions in Ouagadougou, Burkina Faso

Ouédraogo¹,

Zohoncon²,

Traoré

et al. 2016

OJOG

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Aims: We aimed at identifying the high-risk HPV genotypes associated with high-grade dysplastic cervical lesions in Burkina Faso. The available vaccines to Burkina Faso only protect against two high risk HPV genotypes: HPV 16 and 18. Are the genotypes identified in the high-grade precancerous lesions in this survey covered by the available vaccines? Methods: The detection and genotyping of high-risk HPV have been conducted based on 118 formalin-fixed and paraffin-embedded archived tissues using the "HPV Genotypes 14 Real-TM Quant" (Sacace biotechnologies ® , Italy) kit allowing for the detection of fourteen high-risk HPV genotypes: HPV 16, 31, 18, 39, 45, 59, 33, 35, 56, 68, 51, 52, 58 and 66. Results: The prevalence of high-risk HPV infections was 48.8% based on the appropriate PCR results (21/43). The most common HPV genotypes were HPV 39 (21.7%), * Corresponding author.C. Ouédraogo et al. 197HPV 35 (13.0%) and HPV 45 (13.0%). Two cases of multiple infections between HPV 39 -45 and HPV 39 -59 have been observed. HPV 16 was not detected in this study. Conclusions: We noted a high prevalence rate for HPV 39, HPV 35 and HPV 45, which are not covered by the commercial vaccines. We also found that the prevalence of HPV 18 was very low in this study and HPV 16 was not detected.

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Section: Discussionmentioning

confidence: 99%

Distribution of High-Risk Human Papillomavirus Genotypes in Precancerous Cervical Lesions in Ouagadougou, Burkina Faso

Ouédraogo¹,

Zohoncon²,

Traoré

et al. 2016

OJOG

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“…None of the previous studies available in the literature described the possible factors that could influence the amplification success. Therefore, the current study made the option of investigating some of the potential interferences in the nucleic acid obtainment process, namely: tissue fragment size, blocks' storage time, used fixative type, different cDNA synthesis primers and different primer sequences, among others [18].…”

Section: Preparing Ffpe Tissue Sections For Nucleic Acids Extractionmentioning

confidence: 99%

“…It consisted of using 1 ml phosphate buffer saline (PBS) with 5 min incubation at room temperature, followed by full speed centrifugation for 5 min in the R-5418 microfuge (Eppendorf AG, Hamburg, Germany). This procedure is done to remove possible fixative residues that could work as PCR inhibitors [18]. Next, the same process was used in the DNA extraction performed by our team.…”

Section: Preparing Ffpe Tissue Sections For Nucleic Acids Extractionmentioning

confidence: 99%

“…The results show that the PBS washing step inclusion in the samples' extraction preparation process led to some statistically significant advantages such as the obtainment of better RNA concentration results (p = 0.00025), even when the same initial amount of tissue was used. In addition, the washing step allowed obtaining better sample purity levels (p = 0.0000001), increasing the samples amplification success (p = 0.018) both in the standard and in the real-time PCR reactions [18].…”

Section: Preparing Ffpe Tissue Sections For Nucleic Acids Extractionmentioning

confidence: 99%

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Nucleic Acids Extraction from Formalin-Fixed and Paraffin-Embedded Tissues

Gouveia¹,

Ferreira²,

Siqueira³

et al. 2016

Nucleic Acids - From Basic Aspects to Laboratory Tools

Self Cite

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Formalin-fixed paraffin-embedded FFPE tissues are an important sample source for retrospective studies. Despite its ability to preserve proteins and cell morphology, formalin hinders molecular biology tests since it fragments and chemically modifies nucleic acids, especially RNA. Although several studies describe techniques that allow extracting nucleic acids from FFPE tissues, so far there is no consensus in the literature about the best protocol to be used in this type of material. Thus, the current chapter aims to describe the factors affecting the FFPE tissue nucleic acid extracting process, compare the available protocols and to describe the modifications developed by our group in some protocols. Such modifications enable nucleic acids obtainment in satisfactory quantity and quality for molecular biology studies.

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“…Of these unclassified variants, a percentage is predicted to affect splicing. Specific kits are available to isolate RNA from formalin-fixed paraffin-embedded (FFPE) tissue blocks, and previous studies show that PCR, RT-PCR and even next-generation sequencing (NGS) are possible on these RNA samples [12,13]. RNA analysis on RNA isolated from the FFPE tissue is currently not standardly performed but would enable the analysis of somatic splice site variants.…”

Section: Introductionmentioning

confidence: 99%

RNA analysis of cancer predisposing genes in formalin-fixed paraffin-embedded tissue determines aberrant splicing

et al. 2018

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High-throughput sequencing efforts in molecular tumour diagnostics detect increasing numbers of novel variants, including variants predicted to affect splicing. In silico prediction tools can reliably predict the effect of variant disrupting canonical splice sites; however, experimental validation is required to confirm aberrant splicing. Here, we present RNA analysis performed for 13 canonical splice site variants predicted or known to result in splicing in the cancer predisposition genes MLH1, MSH2, MSH6, APC and BRCA1. Total nucleic acid was successfully isolated for 10 variants from eight formalin-fixed paraffin-embedded (FFPE) tumour tissues and two B-cell lines. Aberrant splicing was confirmed in all six variants known to result in splicing. Of one known variant in the B-cell line, aberrant splicing could only be detected after formalin fixation, which indicated that formalin fixation could possibly inhibit RNA degradation. Aberrant splicing was concluded in three of four predicted splice variants of uncertain significance, supporting their pathogenic effect. With this assay, somatic splice variants can be easily and rapidly analysed, enabling retrospective analysis to support the pathogenicity of variants predicted to result in splicing when only FFPE material is available.

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Comparison of Two Methods of RNA Extraction from Formalin-Fixed Paraffin-Embedded Tissue Specimens

Cited by 23 publications

References 12 publications

Distribution of High-Risk Human Papillomavirus Genotypes in Precancerous Cervical Lesions in Ouagadougou, Burkina Faso

Distribution of High-Risk Human Papillomavirus Genotypes in Precancerous Cervical Lesions in Ouagadougou, Burkina Faso

Nucleic Acids Extraction from Formalin-Fixed and Paraffin-Embedded Tissues

RNA analysis of cancer predisposing genes in formalin-fixed paraffin-embedded tissue determines aberrant splicing

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