Isabella Dotti

Effects of formalin, methacarn, and fineFIX fixatives on RNA preservation.

Formalin-fixed tissues represent the most abundant clinical material for retrospective studies. However, formalin highly affects macromolecules, impairing their extraction and analysis. In this study, the suitability of some potential substitutes of formalin for RNA-based applications has been considered. Conventional formalin was compared with methacarn and the commercial FineFIX. Their impact on overall RNA preservation was investigated in a cell line-based model fixed during a time course treatment and in a series of fixed human tissues. RNA yield was detected by Nanodrop; ribosomal RNA (rRNA) integrity by electrophoresis and the Agilent Bioanalyzer; messenger RNA (mRNA) integrity by Northern blot and endpoint reverse transcription-polymerase chain reaction; and mRNA amount by real-time polymerase chain reaction. In the cell line model, formalin fixation showed time-dependent detrimental effects on overall RNA preservation. Methacarn and FineFIX were more conservative on both rRNA and mRNA preservation and their impact was time-independent. In tissues, high rRNA degradation levels were found in all fixed specimens, contrasting with the results found in the cells. Conversely, the effects of the fixatives on mRNA integrity reflected the observations shown in the cell line model. In methacarn-fixed samples mRNA amount was also preserved, whereas in formalin and FineFIX-fixed samples it was notably lower when compared with the fresh frozen control. Alcohol-based fixatives are a good solution for long-term fixation of both cytologic and tissue samples by virtue of their time-independent effects on mRNA preservation. In fixed tissue samples, however, the potential effects of preanalytical tissue-related factors should be considered when performing mRNA quantitative analysis.

Prevalence of Different Subtypes of Serrated Polyps and Risk of Synchronous Advanced Colorectal Neoplasia in Average-Risk Population Undergoing First-Time Colonoscopy

OBJECTIVES:A growing body of evidence indicates that patients with sessile serrated adenoma/polyp (SSA/P) and traditional serrated adenoma (TSA) are at risk for subsequent malignancy. Despite increasing knowledge on histological categorization of serrated polyps (SPs) data are lacking on the actual prevalence and the association of each SP subtype with advanced colorectal neoplasia.METHODS:We prospectively determined the prevalence of different SP subtypes and evaluate the association with synchronous advanced neoplasia in asymptomatic average-risk subjects undergoing first-time colonoscopy. All retrieved polyps were examined by two independent pathologists. Serrated lesions were classified into hyperplastic polyps (HP), SSA/P (without and with cytological dysplasia, SSA/P/DIS), and TSA, and were screened for BRAF and K-ras mutations.RESULTS:Among 258 polyps detected in 985 subjects, the proportion of SSA/P and TSA was 8.9% and 1.9% with an overall prevalence of 2.3% and 0.6%, respectively. SSA/Ps were small without significant difference in their location between proximal and distal colon; TSA were predominantly left-sided. BRAF mutation was common in SSA/Ps and K-ras mutation was present in all TSA. Independent predictors of advanced neoplasia were male sex (odds ratio (OR)=2.0, 95% confidence interval (CI) 1.0–4.0), increasing age (OR=4.5, 95% CI 1.5–13.4 for 50–69 years and OR=9.9, 95% CI 3.1–31.5 for >70 years), current smoking (OR=2.0, 95% CI 1.3–6.8), >3 tubular adenoma (OR=3.6, 95% CI 1.9–6.4), and SSA/P (OR=6.0, 95% CI 1.9–19.5).CONCLUSIONS:The substantial prevalence of BRAF-mutated SSA/P and the independent association with synchronous advanced colorectal neoplasia in asymptomatic average-risk subjects support the overall impact of the serrated pathway on colorectal cancer (CRC) risk in general population. The endoscopic characteristics of SSA/P emphasize the need of high-quality colonoscopy as a key factor for an effective CRC screening program.

Higher random oligo concentration improves reverse transcription yield of cDNA from bioptic tissues and quantitative RT-PCR reliability.

Real time quantitative reverse transcription-PCR (qRT-PCR) is the most sensitive technique for detection and quantification of mRNA targets. Reliable quantification of gene expression in formalin-fixed, paraffin-embedded tissues (FFPE), however, has been subjected to serious limitations so far, mainly due to the fragmentation of RNA transcripts. We tried to improve the sensitivity and reliability of mRNA quantification in FFPE by boosting the reverse transcription (RT) step, that is neglected in most of the protocol analysis, but that represents the first confounding event in a quantitative analysis. For this purpose, we compared yield, reproducibility and linearity of RTs performed with random hexamers, random pentadecamers, or a mixture of antisense specific primers in presence of either Moloney murine leukemia virus (MmLV) or the avian myeloblastosis virus (AMV) enzymes. Random primers were tested at two concentrations, 0.14 and 3.35 nmol/reaction. Our qRT-PCR results indicate an improvement of RT yield when using the highest concentration of random oligos with MmLV (from -1.4 to -4.1 C(t)s) in comparison to the lowest concentration. Moreover, more reliable standard curves and therefore, efficiencies were obtained. RT reactions performed with specific primers and AMV were those with the highest yield, but efficiencies were unreliable, due to the RT enzyme-driven PCR inhibition. Random priming at the 3.35 nmol/reaction concentration seems to be the most convenient strategy in assays using RNA obtained from FFPE tissues.

Quantitative Real-Time RT-PCR

Quantitative real-time PCR is a sensitive tool for quantitative expression analysis of RNA isolated from clinical samples and is increasingly being utilized in novel clinical diagnostic assays. It is an essential tool for the detection of degraded RNA, as that extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Although it has unquestionable potential for expression analysis in archival case studies, there are still some biological and technical problems associated with its use, which can be partly overcome by a standardization of the protocols. The first part of this chapter provides a detailed description of the most commonly used real-time-based chemistries, SybrGreen and TaqMan, focusing especially on their advantages when applied in FFPE material. Specific suggestions have been included in order to improve the reliability of the expression results. The second part of the chapter is dedicated to the description of general guidelines that should be followed to optimize the design of quantitative real-time PCR assays. They have been adapted to RNA isolated from FFPE but should always be taken into account in any gene expression analysis to ensure its accuracy and reproducibility.

Formalin-Free Fixatives

Archival formalin-fixed and paraffin-embedded (FFPE) tissues that represent the standard material used for histologic examination have recently become a valuable source for macromolecule extraction and analysis. Conventional formalin fixation, however, remarkably reduces the quality and quantity of extracted macromolecules, thus affecting the reliability of the experimental results. Moreover, there is increasing evidence that formalin represents a hazardous chemical component that requires specific precautions when used. In the meantime, several alternative fixatives have been proposed to overcome the limitations of using formalin. Among them are alcoholic and nonalcoholic solutions, home-made fixatives and commercial fixatives. In this chapter, the main characteristics of formalin-free fixatives are illustrated, focusing especially on their advantages in terms of morphological preservation, feasibility in immunohistochemical analysis, and macromolecule preservation.

DNA Sequencing from PCR Products

This chapter provides a procedure and some advice for direct sequencing of PCR products from DNA extracted from formalin fixed and paraffin embedded (FFPE) tissues. A section in detail is dedicated to the analysis of the electropherograms. Generally, mutational analysis requires the extraction of DNA from FFPE, PCR amplification and PCR product purification and sequencing. The chapter is dedicated particularly to the analysis of somatic mutations in tumour tissues specifying the importance of analyzing a limited amount of non-mutated DNA component in order to obtain good quality and analyzable electropherograms.

Expression of the Endocannabinoid Receptor 1 in Human Stroke: An Autoptic Study

OBJECTIVE Stroke is one of the leading causes of disability and death in the world. The endocannabinoid (eCB) system is upregulated in several neurological diseases including stroke. A previous animal study demonstrated an increased expression of the endocannabinoid receptor 1 (CB1R) in the penumbra area surrounding the ischemic core, suggesting a crucial role in inflammation/reperfusion after stroke. Regarding the localization of CB1/CB2 receptors, animal studies showed that cortical neurons, activated microglia, and astroglia are involved. Our aim was to evaluate the cerebral expression of CB1R in the ischemic brain areas of 9 patients who died due to acute cerebral infarction in the middle cerebral artery territory. METHODS The cerebral autoptic tissue was collected within 48 hours since death. Ischemic and contralateral normal-appearing areas were identified. After tissue preprocessing, 4-µm-thick cerebral sections were incubated with the primary CB1R antibodies (Cayman Chemical Company, Ann Arbor, MI). Thereafter, all cerebral sections were hematoxylin treated. In each section, the total cell number and CB1R-positive cells were counted and the CB1R-positive cell count ratio was calculated. For statistical analysis, Students t-test was used. RESULTS In normal tissue, CB1R-positive neurons were the majority; a few non-neuronal cells expressed CB1R. In the ischemic areas, a few neurons were detectable. A significant increase in total CB1R staining was found in the ischemic regions compared to contralateral areas. CONCLUSIONS We found an increase in CB1R expression in the ischemic region (neuronal and non-neuronal cell staining), suggesting the inflammatory reaction to the ischemic insult. Whether such response might mediate neuroprotective actions or excitotoxicity-related detrimental effects is still unclear.

DNase Treatment of RNA

The presence of contaminating genomic DNA (gDNA) in RNA preparations is a frequent cause of false positives in RT-PCR-based assays aimed at gene expression analysis. Sometimes this phenomenon cannot be avoided even when specific precautions in the assay design are taken (e.g. design of intron-spanning primers), for example, when the target mRNA presents pseudogenes at the DNA level. For these reasons, the inclusion of a DNase digestion step is often necessary. In this chapter two alternative methods for RNA treatment with DNase are described. The choice of the most suitable method is largely dependent on the availability of starting RNA. The conventional DNase treatment requires a step of phenol/chloroform. This method is useful when an RNA solution is not pure and large RNA amounts are available, since this treatment causes about 50% RNA loss. The DNase digestion followed by enzyme heat inactivation is particularly suitable when an RNA starting quantity is very low because, theoretically, no further RNA is lost during heat treatment. This method may be very useful when an RNA has been extracted from small biopsies or cytologic specimens.

Quantification of Nucleic Acids

Several procedures are currently available for the quantification of DNA and RNA. However, not all these methods are equally useful for the accurate measurement of nucleic acid concentration extracted from fixed and paraffin-embedded samples. This is frequently due to the high degradation levels and low yields of extracted macromolecules, and to the presence of tissue contaminants that may or overestimate the quantity of DNA/RNA suitable for downstream analysis. The methods described in this chapter have been divided into two main categories, according to whether measurement is based on spectrophotometric measurement or on reading in the presence of a fluorescent dye. Each procedure has been illustrated referring to its advantages and/or limitations when used for quantification of nucleic acids extracted from archival samples. This section is focused especially on RNA measurement, for which the majority of the methods relies on detection of ribosomal RNA as an estimated measure of target mRNA quantity. A further section is dedicated to the absolute quantification of specific target sequences by means of the real-time-based amplification that results, particularly suitable when the starting material is extremely low.

General Protocol for Dot-Blot

This chapter provides the general workflow to simply entrap nucleic acids (in the specific case DNA) onto a Nylon membrane by dot-blot technique. The described method is a simple and fast procedure to transfer a known amount of sample onto an inherent support, such as a Nylon membrane. The quantity of the specific target could be then determined by probe hybridization.