PrimerDesign Ltd geNorm TM Houkeeping Gene Selection Kit
with PerfectProbe TM
For the establishment of the optimal normalising genes
Using PerfectProbe TM detection chemistry
Contents
Introduction3 Kit Contents4 Reagents and Equipment to Be Supplied by Ur4 Installing geNorm5 Licensing Agreement and Limitations of u5 PrimerDesign Satisfaction Guarantee5 Quality Control5 Bench-side Protocol6 Amplification Protocol8 geNorm Analysis9 Interpreting geNorm Output Charts.11 FAQ's13
Introduction
For accurate gene expression measurements, it is esntial to normali results from your quantitative real-time PCR experiments to a fixed reference; one that is not affected by your experimental conditions. Although normalising to a constitutively expresd houkeeping gene is the most common method, there is no universal reference gene that is constant in all experimental situations.
女人大For example, reference gene expression levels may vary by:
•The tissue origin of your sample. e.g. brain vs. heart.
•The dia origin of your sample. e.g. carcinoma vs. healthy tissue.
•Your experimental parameters. e.g. stimulated cells vs. non-stimulated.
If your lected reference gene expression is variable, then normalising to this gene will verely limit the accuracy and nsitivity of your assay. Therefore it is necessary to find the ideal reference genes for YOUR particular experimental system and to establish that the genes are indeed not regulated.
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The PrimerDesi gn geNorm kits each contain a panel of 6 or 12 candidate reference genes.
The expression of the genes can be measured by quantitative real-time PCR and the data analyd by the geNorm software.容易饿
The analysis will reveal the best reference gene for accurate normalisation in your experimental system by ranking the candidate reference genes according to their expression stability.
Having ud geNorm to lect the best normalising genes for your experiment, PrimerDesign can provide ready to u high quality normalising gene detection kits for your next experiments.
Kit Contents
•Lyophilid primer and probe ts for 6 or 12 reference genes (BROWN) Sufficient reagents are supplied for 200 20µl reactions per gene.
•RNA/DNA free water (WHITE)
•CD-ROM containing geNorm VBA applet for Microsoft Excel
Also included on the CD-ROM is a detailed geNorm ur manual in PDF format and annotated Excel files for data input.
Reagents and Equipment to Be Supplied by Ur •Real-Time PCR instrument
•Mastermix or mastermix components
This kit is designed to work well with all commercially available Mastermixes. However, we recommend the u of PrimerDesign 2x Precision TM Mastermix.
•Pipettors and Tips
•Vortex and centrifuge
•Sample cDNA
The quality of cDNA will directly affect the quality of data generated using this kit. 10 cDNA samples is the minimum recommended for geNorm analysis. When asssing expression stability in different
高中毕业证书模板groups (e.g. diad vs. healthy) make sure that you u the same number of samples from each group in order to avoid bias towards the group with the largest number of samples
河南的大学•PC running windows XP or 2000 and Microsoft Excel version 2000, XP or 2003 Kit Storage
补假This PrimerDesign kit should be stored at -20ºC on arrival. Freeze/thawing cycles should be kept to a minimum once resuspended. Under the conditions reagents are stable for six months from date of resuspension.
阳光长跑Installing geNorm
Unzip the geNorm_3.4.zip file. After unzipping, a geNorm directory is created, which contains the geNorm.xls applet, and an InputData directory and OutputData directory.
The InputData directory contains a demo data file (fibroblast.xls described in Vandesompele et al., 2002, Genome Biology), and the OutputData directory contains a detailed ur manual as a PDF file).
Licensing Agreement and Limitations of u The geNorm VBA applet for Microsoft Excel is freely available (on request) from the author (medgen.ugent.be/~jvdesomp/genorm/) to all tho invol
ved in not-for-profit academic rearch.
Commercial u of geNorm software is available only via a limited licence when ud in conjunction with PrimerDesign kits.
椅子英文PCR is covered by veral patents owned by Hoffman-Roche Inc and Hoffman-LaRoche, Ltd. Purcha of PrimerDesign kits does not include or provide licence with respect to any patents owned by Hoffman-La Roche or others.
PrimerDesign Satisfaction Guarantee
PrimerDesign takes pride in the quality of all its products. Should this product fail to perform satisfactorily when ud according to the protocols in this manual, PrimerDesign will replace the item free of charge.
Quality Control
As part of our routine quality assurance programme, all PrimerDesign products are monitored to ensure the highest levels of performance and reliability.
