Applied Biosystems 7900HT User Manual

DRAFT
September 1, 2004 11:39 am, CH_Real-Time.fm
Chapter 6 Analyzing Real-Time Data
6-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
Real-Time Runs on the 7900HT Instrument
Real-Time Runs
Real-time is the term used to describe the category of sequence detection runs in
which the Applied Biosystems 7900HT Fast Real-Time PCR System is used to
measure the fluorescence of a biological sample during thermal cycling. In contrast
to end-point runs, real-time experiments can be used to achieve both qualitative and
quantitative measurements. Real-time analysis can be used in combination with
either TaqMan
®
or SYBR
®
Green 1 double-stranded DNA binding dye reagents for a
variety of purposes including quantitative PCR and dissociation curve analysis.
Quantitative
RT-PCR
Quantitative RT-PCR is a method used to measure small quantities of ribonucleic
acid sequences isolated from biological samples. Typical biological samples include
cells, tissues, and fluids. During the RT step, reverse transcription of target RNA
produces corresponding complementary DNA (cDNA) sequences. During the
subsequent PCR, the initial concentration of target cDNA is quantified by amplifying
it to a detectable level. The two types of quantitative RT-PCR supported by the
7900HT instrument, absolute and relative, are described in detail below.
Absolute
Quantification
The objective of an absolute quantification experiment is to accurately determine the
absolute quantity of a single nucleic acid target sequence within an unknown sample.
The results of an absolute quantification experiment are reported in the same units of
measure as the standard used to make them.
Relative
Quantification
The objective of relative quantification is to determine the quantity of a single
nucleic acid target sequence within an unknown sample relative to the same sequence
within a calibrator sample. Relative quantification of target gene expression is
calculated from Applied Biosystems 7900HT Fast Real-Time PCR System data
using one of the following methods:
About the Standard Curve Method
The Standard Curve Method constructs a standard curve similar to that used in
absolute quantification experiments. However, because the goal of relative
quantification is to establish a fractional relationship, data produced by relative
quantification standards is used differently. For all experimental samples, target
quantity is determined from the standard curve and divided by the target quantity of a
calibrator sample. Because the unit from the standard curve drops out, only the
relative dilutions of the standard are necessary to determine relative quantities.
About the Comparative C
T
Method
The SDS software calculates relative levels of gene expression using the
Comparative C
T
Method of data analysis. The Comparative C
T
Method is similar to
the Standard Curve Method except that it uses arithmetic formulas to achieve the
same results for relative quantification. Instead of a standard curve, the Comparative
C
T
Method calculates relative gene expression using the equation:
Relative Quantity = 2
–∆∆C
T

Applied Biosystems 7900HT Specifications

General

Brand	Applied Biosystems
Model	7900HT
Category	Laboratory Equipment
Language	English