All‐in‐One
ForquantitativedetectionofmaturemiRNA
Cat.No.AOMD‐Q020(20RTand200qPCRreactions)
Cat.No.AOMD‐Q050(50RTand500qPCRreactions)
UsedincombinationwiththeAll‐In‐One™miRNAqPCRPrimers
™
miRNAqRT‐PCRDetectionKit
UserManualI
GeneCopoeia,Inc.
9620MedicalCenterDrive,#101
Rockville,MD20850
USA
301‐762‐0888
866‐360‐9531
inquiry@genecopoeia.com
www.genecopoeia.com
© 2009 GeneCopoeia, Inc.
All-in-One™ miRNA qRT-PCR Detection Kit User Manual
USER MANUAL I
™
All-in-One
miRNA qRT-PCR Detection Kit
I. Introduction and Principle
II. Related Products
III. Contents and Storage
IV. Preparation
V. Procedure
VI. Examples
VII. Trouble Shooting Guide
VIII. Limited Use License and Warranty
I. Introduction and Principle
Small, non-coding miRNAs are widely present in eukaryotes. They consist of about 22 nucleotides that
control many important physiological processes in cell development and differentiation. Different miRNAs
express differently at different developmental stages and different tissues. Therefore, the quantitative
assaying of miRNAs is important in both basic and applied research.
The All-in-One™ miRNA qRT-PCR Detection Kit uses real-time PCR technology to quantitatively measure
miRNAs. The experimental procedure includes three major steps (Figure 1)
1) Single-step cDNA Synthesis - Poly A polymerase is used to add poly-A tails to the 3’ end of
miRNAs
2) cDNA Synthesis - At the same time M-MLV RTase and a unique Oligo-dT Adaptor primer reverse
transcribes the poly A miRNAs (The Universal Adaptor PCR primer in combination with a miRNAspecific primer allows detection of specific miRNA)
3) qPCR - The All-in-One qPCR Mix containing SYBR
transcribed miRNA (The miRNA-specific forward primer is used with the Universal Adaptor primer)
Compared to traditional hybridization-based miRNA detection methods such as Northern blot analysis, the
method provided by the All-in-One qRT-PCR kit is faster, more specific and sensitive and use less sample
material.
Advantages of the All-in-One miRNA qRT-PCR Detection Kit
• Provides efficient reverse transcription of miRNAs into cDNA in a single step
• Delivers a precise quantitative and accurate measurement of miRNA expression profiles
• Differentiates between mature and precursor miRNA
• Co-developed with validated primers, miRNA clones and other tools used for functional studies of
miRNA
®
Green specifically detects the reverse
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All-in-One™ miRNA qRT-PCR Detection Kit User Manual
Figure 1. A graphic representation of the steps involved in the All-in-One qRT-PCR miRNA Detection
Kit.
II. Related Products
GeneCopoeia offers comprehensive solutions for studying miRNAs. A careful process of co-development
ensures that they work well together and provide robust and reproducible results.
GeneCopoeia Description
miExpress™ Precursor miRNA
Expression Clones
miTarget™ miRNA Target Validation
Expression Clones
OmicsLink™ Expression-Ready ORF
cDNA Clones
750 human
450 mouse
270 rat
25,000 human
25,000 mouse
20,000 human
15,000 mouse
Study the regulatory and functional effect of
miRNAs on corresponding genes and proteins
Cross validate data using luciferase reporter
genes
Perform gain-of-function studies with
expression-ready clones
All-in-One™ miRNA qRT-PCR Detection
Kits
All-in-One™ miRNA qPCR Primers
Endofectin™ Transfection Reagents
Accurately quantify miRNA expression
Validated for robust, reproducible and reliable
quantitation of miRNA activity
Optimized for specific
cell types
Transfect efficiently and with low toxicity for
reliable and reproducible results
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All-in-One™ miRNA qRT-PCR Detection Kit User Manual
III. Contents and Storage
Contents and storage recommendations for the All-in-One miRNA qRT-PCR Detection Kits
(Cat. Nos. AOMD-Q020 and AOMD-Q050) are provided in the following table.
Contents Quantity Storage temperature/ conditions
–20°C (Stable for at least 12 months)
2.5 U/µl
Poly A Polymerase
RTase Mix
20 µl
50 µl
20 µl
50 µl
Alternatively, the solution can also be
stored at -80°C in aliquots. Avoid
repeated freezing/ thawing.
–20°C (Stable for at least 12 months)
Alternatively, the solution can also be
stored at -80°C in aliquots. Avoid
repeated freezing/ thawing.
5X Reaction Buffer
dd H2O
(RNase and DNase free)
All-in-One qPCR Mix
50X ROX Reference Dye 80 µl
100 µM
Universal Adaptor PCR
Primer
T
= 64.5
m
GC content = 50%
100 µl
250 µl
1 ml
1 ml
2 X 1 ml
5 X 1 ml
200 µl
20 µl
50 µl
–20°C (Stable for at least 12 months)
Alternatively, the solution can also be
stored at -80°C in aliquots. Avoid
repeated freezing/ thawing.
–20°C (Stable for at least 12 months)
Alternatively, the solution can also be
stored at -80 °C in aliquots. Avoid
repeated freezing/ thawing.
–20°C (Stable for at least 12 months)
Alternatively, the solution can also be
stored at -80 °C in aliquots. Avoid
repeated freezing/ thawing.
–20°C (Stable for at least 12 months)
Alternatively, the solution can also be
stored at -80 °C in aliquots. Avoid
repeated freezing/ thawing.
–20°C (Stable for at least 12 months)
Alternatively, the solution can also be
stored at -80 °C in aliquots. Avoid
repeated freezing/ thawing.
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