For research purposes only. Not for use in diagnostic procedures for clinical purposes. Ready-to-use amplification primer mix for RT-PCR using the LightCycler™ Instrument Human Dex-I (dexamethasone-induced ; MYLE) Note: After Thawing keep on ice! material Vial Label Content and use contents
ready-to-use primer mix for target specific
Yellow cap
amplification using the LightCycler™FastStart
Preparation
achieved with 1µg total RNA isolated with
contains optimal MgCl2 concentration andamplification primer pair
amplification standard for approximately 34000
Strand cDNA Synthesis Kit (AMV) (Roche). ! The resulting cDNA has to be diluted to a final volume of 200-500 µl with PCR- Green cap grade water
contains a cDNA mix from several humanhematopoietic cell lines
Application
Quantitative evaluation of gene expression
White cap Assay time
Set up the PCR amplification 15 min Additional
1st Strand cDNA Synthesis Kit for RT-PCR (Roche Cat. # 1 483 188)
equipment
LightCycler™ FastStart Master SybrGreen I (Roche Cat. # 3 003 230)
LightCycler™ PCR run 50 min
LightCycler™ Instrument (Roche Cat. # 2 011 468)
LightCycler™ Primer Set Housekeeping genes (Search GmbH)
reagents Number of required
The LightCycler™-Primer Set is tested using
The LightCycler™-Primer Set allows to perform quantitative
RT-PCR using the LightCycler™ instrument. An optimized
primer pair has been selected for specific amplification oftargets. The amplicon is detected by fluorescence using the
Kit storage/
The unopened kit is stable at –20˚C 12 month
double-stranded DNA binding dye Sybr®Green I. stability Specificity
The LightCycler™-Primer Set “Dex-I” isspecific for the sequence of human Dex-I. The primers were selected in the first exon of
the gene based on their sensitivity, since dueto the presence of a pseudogene onchromosome 16, genomic DNA will bedetected also with intron overspanningprimers. However, no genomic signal will begenerated if RNA or mRNA is generated asdirected (DNAse treatment). If the samplequality is poor or unknown a no-RT controlreaction is strongly recommended. Amplification Introduction
A fragment of the human Dex-IcDNA sequence is amplified and
Parameter Additional reagents required Thawing the solutions Melting Curve Analysis
LightCycler™ FastStart vial 1a/b Parameter It is recommended to define the experimental protocol before preparing the solutions Experimental Protocol
Program 1: Denaturation ofthe template and activation of
Parameter Denaturation Parameter Preparation of Depending on the total number of Typical results the master Introduction
five additional reactions(Standard). Quantification
in the QC sheet were obtained byperforming the describedprocedure with the enclosed
Step Action
standards and control cDNA. Thefluorescence values versus cycle
Prepare a fresh dilution series of the standard
In a 1.5 ml light protected reaction tube onice, add the following components in theorder mentioned below:
Melting curve Component
LightCycler™ Primer Set (yellow cap) 2 µl Total Volume
• Pipet 10 µl PCR mix into the precooled
• Add 10 µl of cDNA template
• Pipet 10 µl of PCR mix into 4 precooled
• Add 10 µl of undiluted and of the freshly
Seal each capillary with a stopper and place
the adaptors, containing the capillary, into a
benchtop microcentrifuge. Centrifuge at 2000
Place capillaries in the rotor of theLightCycler™ Instrument.
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Heidelberg
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Preparation of Depending on the total number of
