Jak2 qPCR Detection Kit-24rxn

JAK2 V617F Detection Kit
For Real-Time PCR
Cat. No. PQ3641 Quantity: 25 Reactions Storage: -20 °C

For Use With:
 Applied Biosystems® – 7500/7500 Fast, StepOne/ StepOne Plus
 Qiagen Rotor-Gene® Q
 LightCycler® 480
 QuantStudio™ 5/6
 Applied Biosystems® – 7900HT
 Qiagen Rotor-Gene® 3000, 6000
 Cobas® 4800, Cobas® z480
 Bio-Rad® CFX96
Intended use
The chronic myeloproliferative Philadelphia-negative diseases (CMPD) are a group of stem hemopoietic
disorders including the polycythemia vera (PV), the essential thrombocytosis (TE) and the idiopathic
myelofibrosis (CIMF). In 2005 the presence of a somatic mutation (V617F) of gene Janus Kinase 2 (JAK2),
coding for an important protein for signal transduction inducted by hemopoietic growth factors, in
patients CMPD Ph negative has been shown; it phosphorylates different cytoplasmic molecules,
particularly the STAT (Signal Transducers and Activators of Transcription). The protein JAK2 belongs to
the Janus kinases family; these proteins are composed by seven regions: JH1-JH7. Particularly, JH1 is
the kinase action region. JH2 is pseudo-kinase domain, important for JH1 catalytic activity and involved
in the inhibitory regulation of that activity. The mutation V617F, is caused by the nucleotide substitution
G>T at nucleotide 1849 in the exon 14, inducing the substitution of the valine amino acid with
phenylalanine at codon 617 (GTC>TTC). This mutation involves a portion of the pseudo-kinase JH2 of
JAK2, important in controlling JH1 inhibitory activity. This is an acquired somatic mutation, that can be
found only in myeloid cells (erythroid line, granulocyte-macrophage line, megakaryocyte line) in the
heterozygous and homozygous state. The mutation is called “gain-of- function mutation” because it
determines a constituent activation of the JAK-STAT pathway, able to give a proliferative advantage and
cytokine-independent growth of the hemopoietic cells.
The mutation V617F has been often noticed in patients with:
 Polycythemia vera (65-97%), 20% more than other homozygous patients.
 Essential thrombocytosis (23-57%), generally present in heterozygous form.
 Myelofibrosis (35-95%)
Less frequently V617F has been found in other diseases such as chronic myelomonocytic leukemia, the
myelodysplastic diseases (MDS), systemic Masto cytosis (SM), the chronic neutrophilic leukemia, the
hypereosinophilic syndrome and the atypical chronic myeloproliferative diseases. The mutation JAK2-
V617F has never been found in healthy subjects. The mutation’s study can explain the disease and can
be used as a diagnostic and prognostic.
Reagents
Package Contents
The JAK2 V617F product contains the following materials sufficient for 25 reactions.
JAK2-V617F kit contents:
Ŧ The primer mix tube containsprimers/probes for both JAK2-V617F (FAM) and endogenous control gene (HEX).
Reagent preparation (for all instruments)
The reactions are setup in a total volume of 20 µl. Reaction mixes for multiple samples (as well as
control samples) should be pre-mixed as a master mix with 5% excess volume to compensate for
pipetting losses.
Each 25 µl reaction contains the following components:
Component Volume (µl)
2x Master Mix Probe 10
JAK2 V617F Detection Primer/ Probe Mix 7
DNA sample 3
Cycling parameters
Place the plate or strip in the thermal cycler and run the following program:
Temperature Time Cycles Data Collection
95oC 10 min 1
95oC 10 sec
45
60oC 40 sec FAM, HEX
Description No. of Tubes Vol. per Tube (µl)
2x Master Mix Probe 1 250
JAK2-V617F
Detection Primer/Probe MixŦ 1 175
Control Positive 1% JAK2 V617F mutant 1 20
Result Interpretation
Evaluation of Extracted DNA quality and Real-time PCR procedure
Endogenous Control Gene:
Endogenous control
Gene Ct is:
Control
Status
24 ≤ Ct ≤ 30 Sufficient DNA
24 > Ct
Overloaded reaction. May observe non-specific signal. Re-test sample with less DNA
to bring HEX within acceptable range.
Ct >30 Low DNA concentration. A rerun with more DNA is recommended.
Interpretation of Target Gene JAK2 V617F
ΔCt:
Ct (FAM) Target Gene JAK2 V617F – Ct (HEX) Endogenous Control Gene
Mutation
Status
ΔCt > 6 Negative
2 < ΔCt ≤ 6 Heterozygote Positive
ΔCt ≤ 2 Homozygote Positive
Limit of Detection
The reaction is setup for detection of 1% mutation JAK2 V617F