Quantification of Cytokine mRNAs in Human Myocardial
Biopsy Samples by Real-Time Quantitative PCR
Technology Using the LightCycier Instrument
XI ZHU, GEORG BAUMGARTEN, FENG WANG, ZIAD DIBBS,
ABHINAV DIWAN, GUILLERMO TORRE-AMIONE, DOUGLAS 1. MANN,
NATARAJAN SIVASUBRAMANIAN*
Introduction
Understanding the pathophysiology of human heart failure [1-3] necessitates the
sensitive measurement of gene expression in human myocardium. Estimating the
quantities of mRNAs in an explanted failing human heart sample after heart
transplantation is a relatively easy task because of the availability of large-sized
tissue samples. However, when the availability of tissue samples is very low, especially from patients who are undergoing therapeutic treatment [4], estimating the
quantities of mRNAs requires a highly sensitive method of measurement. In this
study, we have developed a highly sensitive method for the quantification of
cytokine mRNAs in human myocardial biopsy tissue samples as low as 2-3 mg.
This method involves isolation of total RNA from frozen biopsy tissue samples,
capturing and immobilizing poly-A RNA, reverse transcription of RNA to obtain
single-stranded complementary DNA, and quantification of cytokine mRNAs
using a real-time quantitative polymerase chain reaction method.
Materials
Biometra Gradient Thermocycler (G6ttingen, Germany)
Equipment
LightCycler and data analysis software version 3.0 (Roche Diagnostics, Indianapolis, IN, USA)
Beckman 540 Spectrophotometer
Dataminder 3.2 software for primer design (Karen Usdin, NIDDK, NIH, MA, USA)
Polytron 3000 (Brinkmann Instruments, Inc., Westbury, NY, USA)
Oligonucleotides (InVitrogen, San Diego, CA, USA)
Reagents
Qiagen plasmid purification kits (Qiagen, Germany)
RNA STAT-60 (Tel-Test Inc., USA)
TRl zOL Reagent (InVitrogen)
* N. Sivasubramanian (~) (e-mail: nats@bcm.tmc.edu)
Winters Center for Heart Failure Research, Cardiology Section of the Department
of Medicine, Veterans Administration Medical Center, Baylor College of Medicine,
Houston, TX 77030, USA
•
Biopsy Samples by Real-Time Quantitative PCR
Technology Using the LightCycier Instrument
XI ZHU, GEORG BAUMGARTEN, FENG WANG, ZIAD DIBBS,
ABHINAV DIWAN, GUILLERMO TORRE-AMIONE, DOUGLAS 1. MANN,
NATARAJAN SIVASUBRAMANIAN*
Introduction
Understanding the pathophysiology of human heart failure [1-3] necessitates the
sensitive measurement of gene expression in human myocardium. Estimating the
quantities of mRNAs in an explanted failing human heart sample after heart
transplantation is a relatively easy task because of the availability of large-sized
tissue samples. However, when the availability of tissue samples is very low, especially from patients who are undergoing therapeutic treatment [4], estimating the
quantities of mRNAs requires a highly sensitive method of measurement. In this
study, we have developed a highly sensitive method for the quantification of
cytokine mRNAs in human myocardial biopsy tissue samples as low as 2-3 mg.
This method involves isolation of total RNA from frozen biopsy tissue samples,
capturing and immobilizing poly-A RNA, reverse transcription of RNA to obtain
single-stranded complementary DNA, and quantification of cytokine mRNAs
using a real-time quantitative polymerase chain reaction method.
Materials
Biometra Gradient Thermocycler (G6ttingen, Germany)
Equipment
LightCycler and data analysis software version 3.0 (Roche Diagnostics, Indianapolis, IN, USA)
Beckman 540 Spectrophotometer
Dataminder 3.2 software for primer design (Karen Usdin, NIDDK, NIH, MA, USA)
Polytron 3000 (Brinkmann Instruments, Inc., Westbury, NY, USA)
Oligonucleotides (InVitrogen, San Diego, CA, USA)
Reagents
Qiagen plasmid purification kits (Qiagen, Germany)
RNA STAT-60 (Tel-Test Inc., USA)
TRl zOL Reagent (InVitrogen)
* N. Sivasubramanian (~) (e-mail: nats@bcm.tmc.edu)
Winters Center for Heart Failure Research, Cardiology Section of the Department
of Medicine, Veterans Administration Medical Center, Baylor College of Medicine,
Houston, TX 77030, USA
•