Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods.

نویسندگان

  • T D Schmittgen
  • B A Zakrajsek
  • A G Mills
  • V Gorn
  • M J Singer
  • M W Reed
چکیده

Four quantitative reverse transcription-PCR (RT-PCR) methods were compared to evaluate the time course of mRNA formation and decay. Mouse fibroblasts (NIH 3T3) transfected with the human beta-globin open reading frame/c-myc 3'-untranslated region chimeric gene under control of the c-fos promoter (fos-glo-myc) were used for serum-inducible transcription. The amount of fos-glo-myc mRNA, relative to beta-actin, was measured by quantitative, RT-PCR at various times following the addition of serum to serum-starved fibroblasts transfected with the chimeric gene. Both endpoint (band densitometry and probe hybridization) and real-time (SYBR green and TaqMan) PCR methods were used to assay the identical cDNA. The real-time methods produced a 4- to 5-log dynamic range of amplification, while the dynamic range of the endpoint assays was 1-log. The real-time and probe hybridization assays produced a comparable level of sensitivity that was considerably greater than band densitometry. The coefficient of variation from 22 replicate PCR reactions was 14.2 and 24.0% for the SYBR green and TaqMan detection, respectively, and 44.9 and 45.1% for the band densitometry and probe hybridization assays, respectively. The rank order for the values of r(2) obtained from the linear regression of the first-order mRNA decay plots was SYBR green > TaqMan > probe hybridization > band densitometry. Real-time PCR is more precise and displays a greater dynamic range than endpoint PCR. Among the real-time methods, SYBR green and TaqMan assays produced comparable dynamic range and sensitivity while SYBR green detection was more precise and produced a more linear decay plot than TaqMan detection.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Development and Evaluation of Real-Time Reverse Transcription Polymerase Chain Reaction Test for Quantitative and Qualitative Recognition of H5 Subtype of Avian Influenza Viruses

Avian influenza viruses (AIV) affect a wide range of birds and mammals, cause severe economic damage to the poultry industry, and pose a serious threat to humans. Highly pathogenic avian influenza viruses (HPAI) H5N1 were first identified in Southeast Asia in 1996 and spread to four continents over the following years. The viruses have caused high mortality in chickens and various bird species ...

متن کامل

Diagnosis of Foot-and-Mouth Disease Virus by Real Time Reverse Transcription Polymerase Chain Reaction Assay in Iran

Background and Aims: Accurate and rapid diagnosis is necessary for effective control and prevention of foot-and-mouth disease (FMD). In present study, was evaluated real time reverse transcription-polymerase chain reaction (rRT-PCR) assay along with diagnostic routine methods for the detection of all seven serotypes of FMD virus (FMDV), namely O, C, A, SAT1, 2, 3 and Asia 1 in biological sample...

متن کامل

Effects of feed restriction and dietary fat type on mRNA expression of liver fatty acid-binding protein (L-FABP) in broilers

Background: Liver fatty acid-binding protein (L-FABP) is the main cytosolic binding site for long chain fatty acids in hepatocytes. FABPs enhance the uptake of fatty acids into the cell by increasing their concentration due to decreasing concentration of unbound fatty acids inside the cell. Objectives: The aim of this study was to evaluate the effects of dietary unsaturated to saturated fatty a...

متن کامل

Application of Real-time Polymerase Chain Reaction (RT-PCR)

The real-time polymerase chain reaction (RT-PCR), also called quantitative real-time polymerase chain reaction (QRT-PCR) or kinetic polymerase chain reaction (kPCR), is a technique used to simultaneously quantify and amplify a DNA molecule. It is used to determine whether a specific DNA sequence is present in the sample; and if it is present, the number of copies in the sample. It is the real-t...

متن کامل

Comparison of Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Agglutination Assays in Diagnosis of Brucellosis in Golestan Province, North of Iran

Introduction: Brucellosis is one of the most common zoonotic infections worldwide. The clinical symptoms of brucellosis are similar to a wide range of diseases; hence, reliable diagnostic and laboratory methods are required to identify the causative agent. Iran is an endemic region of brucellosis, and many patients are misdiagnosed due to the nature of the infection. In this study, we aimed to ...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • Analytical biochemistry

دوره 285 2  شماره 

صفحات  -

تاریخ انتشار 2000