sybr-green-i and Liver-Neoplasms

The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR), which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1) DNA extracted from fresh frozen liver tissues (Frozen-DNA); 2) DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA); and 3) DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA). These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA) quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method for quantifying DNA available for PCR.

Topics: Animals; Benzothiazoles; Carcinoma, Hepatocellular; Diamines; DNA; Fixatives; Fluorescent Dyes; Formaldehyde; High-Throughput Nucleotide Sequencing; Humans; Liver; Liver Neoplasms; Organic Chemicals; Paraffin Embedding; Quinolines; Rats; Rats, Long-Evans; Real-Time Polymerase Chain Reaction; Solutions; Tissue Fixation

Due to the fact that mutations and up- or downregulation of genes can lead to the development of cancer, quantitative comparison of relative gene expression in healthy and cancerous tissue can gain valuable insights into tumorigenesis. While the semi-quantitative DNA microarrays are being used to identify differentially expressed genes on a genomic scale, real-time RT-PCR provides a powerful tool for quantitative measurement of gene expression. Presently, it is the most sensitive method available. Here we describe in detail a SYBR GreenI-based assay using the LightCycler instrument to measure the levels of mRNA for the ubiquitin-like protein FAT10 relative to 18S rRNA in human hepatocellular carcinoma tissue. This method can be easily adapted to any tissue (human or mouse, rat, etc.) and any gene.

Topics: Benzothiazoles; Carcinoma, Hepatocellular; Diamines; Gene Expression; Humans; Liver Neoplasms; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Ribosomal, 18S; Ubiquitins

To detect the expression of CD95 and CD95L mRNAs after liver transplantation and investigate the role of CD95 and CD95L in acute liver allograft rejection.. The expressions of CD95 and CD95L mRNAs of peripheral blood lymphocyte from 56 liver allograft recipients were examined using SYBR real-time PCR.. CD95 and CD95L mRNA levels in the recipients with acute rejection were significantly higher than those without rejection (P<0.01), and the elevation occurred about 2 days earlier than that of alanine aminotransferase and aspartate aminotransferase.. CD95 and CD95L are related to acute liver allograft rejection and their mRNA expression level may serve as an indicator for prediction and diagnosis of acute rejection episodes.

Topics: Acute Disease; Adult; Aged; Benzothiazoles; Diamines; Fas Ligand Protein; fas Receptor; Female; Gene Expression; Graft Rejection; Humans; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Lymphocytes; Male; Middle Aged; Organic Chemicals; Polymerase Chain Reaction; Quinolines; RNA, Messenger