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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All-in-One™ miRNA qRT-PCR Detection Kit User Manual
IV. Preparation
Wearing a lab coat, disposable gloves and protective goggles are recommended when handling
chemicals.
RNA Sample Preparation
When working with RNA it is important to avoid RNases in your solutions, consumables and labware. When
preparing your RNA samples, always wear a mask and disposable gloves in all procedures. Follow the
described procedures you are using for RNA extraction carefully. Ready-to-use solutions that are RNasefree can be purchased. Alternatively treat solutions with diethyl pyrocarbonate (DEPC) and then autoclave.
RNases on labware can also be inactivated by DEPC treatment or by baking at 250°C for 3 hours. Use
DEPC to treat all microcentrifuge tubes, pipettes and pipette tips (if no RNase free) and then autoclave to
deactivate RNases. RNase-free consumables are available for purchase from many commercial sources.
Primer Design
The reverse primer called “Universal Adaptor PCR Primer” (T
the All-in-One miRNA qRT-PCR Detection Kit.
You may wish to design and make specific forward primers for your miRNA of interest or order from
GeneCopoeia. Please contact us for further information.
Since the length of miRNAs is generally between 18 ~ 24 nucleotides for some “easy” miRNAs, a forward
primer may be designed directly according to the sequence of the miRNA. However, for some potentially
“difficult” miRNAs (e.g. very high or very low T
tissues (e.g. tissues with high pre-miRNA/pri-miRNA) special primers may need to be designed to optimize
the primer sequence in order to obtain specific amplification and avoid interference from pre- miRNA/primiRNA.
or highly homologous miRNAs) or miRNAs from specific
m
IMPORTANT NOTES:
1. Store kit at –20°C. Avoid storage or leaving reagents at 4°C or room temperature.
2. Mix reagents thoroughly by gently inverting tubes several times avoiding bubbles, and then briefly
centrifuge before use.
3. Following the procedure carefully to avoid contamination with RNases which can rapidly degrade
RNA and lead to inconclusive results.
4. Set up all reactions on ice to reduce risk of RNA degradation.
V. Procedure
1. Reverse transcription of miRNA
a. Thaw template RNA on ice. Thaw 5X RT Buffer and ddH2O (RNase/ DNase free) at room
temperature (15–25°C).
b. Gently mix miRNA reverse transcription reagents by flicking to dissolve all reagents thoroughly.
Briefly centrifuge to collect residual liquid from the sides of the tubes and then place on ice.
c. Prepare miRNA reverse transcriptase reaction solution.
Place RNase-free reaction tubes on ice and then add the following reagents to a final volume of
25 µl.
= 64.5, GC% = 50%) has been provided in
m
5
All-in-One™ miRNA qRT-PCR Detection Kit User Manual
Reagent Volume Quantity
Total RNA
or small-molecule RNA
2.5 U/ µl Poly A Polymerase 1 µl
RTase Mix 1 µl
5X Reaction Buffer 5 µl 1X
dd H2O (RNase-/DNase- -free) To final 25 µl
* Total RNA must contain small-molecule RNA
† The amount of total RNA can be between 1 ng ~ 5 µg. If using purified small-molecule RNA, the
amount can be between 0.1 ng ~ 1 µg.
d. Prepare reverse transcription reaction.
Mix the prepared reaction mix gently, but thoroughly. Incubate at 37°C for 60 minutes after a brief
centrifugation.
Incubate at 85°C for 5 minutes to inactivate the enzyme.
The resulting reverse transcription reaction product should be diluted 5 ~ 50 times with sterile H20
before using for the next qPCR experiment or it can be directly stored at –20°C.
2. Detection of miRNA with qPCR.
a. Dissolve 2X All-in-One qPCR Mix by gently inverting. Briefly centrifuge and place on ice. If required,
dissolve 50X ROX Reference Dye.
b. Dilute the 100µM Universal Adaptor PCR Primer to 2µM with sterile ddH
qPCR experiment.
c. Prepare qRT-PCR solution on ice. See example.
†
2 µg
100 ng
0 before using for the next
2
Reagent Volume Final concentration
2X All-in-One qPCR Mixi
All-in-One miRNA qPCR Primer (2 µM)
ii
2 µl 0.2 µM
10 µl 1X
Universal Adaptor PCR Primer (2 µM) 2 µl 0.2 µM
First-strand cDNA (diluted 1:5)
iii
2 µl
50X ROX Reference DyeiV 0.4 μl 1X
Water (double distilled)
■ Not using ROX Reference Dye 4 μl
■ Using ROX Reference Dye 3.6 μl
Final volume 20 μl
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All-in-One™ miRNA qRT-PCR Detection Kit User Manual
Notes
i. Use the 2X All-in-One qPCR Mix as half of the total reaction volume and adjust other
reagents accordingly. If the total reaction volume is changed, maintain each component in
proper proportion.
ii. Primer concentration should be in the range of 0.2 to 0.4 μM. In general, a PCR reaction
using 0.2 μM primers produces good results
iii. The first-strand cDNA should be diluted before using for the PCR reaction in order to avoid
interference to the qPCR from the reverse transcription system.
iv. ROX Reference Dye is used in Real-Time PCR instruments that require ROX for calibration,
such as the ABI qPCR instrument.
d. Thoroughly mix the qPCR reaction solution, add to PCR tubes, and briefly centrifuge to make sure
that all the reagents are in the bottom of the tubes.
e. The following standard 3-step method for the qPCR reaction is recommended (example adapted
from the iQ5 real-time PCR detection system from Bio-Rad).
Cycles
Steps Temperature Time Detection
1 Initial denaturation 95°C 10 min No
Denaturation 95°C 10 sec No
40
Notes
i. When using SYBR Green dye to monitor the qPCR reaction, a melting curve analysis should
be performed immediately after qPCR cycling. For instructions, consult the documentation
for your qPCR instrument. The following is an example adapted from the iQ5 real-time
detection system from Bio-Rad Laboratories. The conditions for your instrument may differ:
Temperature Range
65°C ～ 95°C 0.5°C/ time 6 sec/ time Yes
30°C
ii. The DNA polymerase used in the 2X All-in-One qPCR Mix is a chemically especially
modified hot-start enzyme. Incubation for 10 minutes at 95°C will sufficiently activate the
enzyme.
iii. Specific properties of a miRNA lead to special properties of the designed primer. Therefore
the annealing temperature needs to be strictly controlled in order to avoid non-specific
Annealing Tm –2°C 20 sec No
Extension 72°C At least 10 sec Yes
Heating Rate Constant Temperature Detection
30 sec No
7
All-in-One™ miRNA qRT-PCR Detection Kit User Manual
amplifications. For validated miRNA primers purchased from GeneCopoeia, please refer to
the optimal conditions for the experiment.
iv. The Oligo-dT Adaptor primer for reverse transcription is 53 nucleotides, therefore the
resulting PCR amplification fragment is about 75bp (assuming the sequence of miRNA is
about 22 nucleotides), which requires at least about 10 seconds extension time. From the
melting temperature of the products the T
value is generally determined to be between
m
75°C ～ 83°C. If the melting temperature exceeds this range, other assaying methods such
as electrophoresis are suggested for the specific properties of the product.
v. The main conditions for the above reactions are for use with the iQ5 qPCR instrument from
Bio-Rad. If a qPCR instrument from another commercial source is used, please reference
the instrument manual and adjust the extension time and melting curve conditions
accordingly.
VI. Examples
a) Example 1: Specificity assay using the All-in-One miRNA qRT-PCR Detection Kit
With 200 ng total RNA mixture from human brain and heart as template, the miRNA qRT-PCR
Detection Kit and the All-in-One miRNA qPCR Primers were used to detect 30 miRNA and an
internal reference snRNA U6. Results from qRT-PCR and electrophoresis showed neither nonspecific amplification products nor primer-dimer formation.
Figure 1. Amplification and melting curves of 31 miRNA and the internal reference snRNA U6, in
which double channel detection was used for the positive control, and single channel detection was
used for the NTC (No Template Control).
8
All-in-One™ miRNA qRT-PCR Detection Kit User Manual
Figure 2. Agarose gel electrophoresis (3% agarose gel) of the amplification products of 31 miRNA
and the internal reference snRNA U6, in which double channel detection was used for the positive
control, and single channel detection was used for the NTC (No Template Control).
b) Example 2: Sensitivity assay using the All-in-One miRNA qRT-PCR Detection Kit
Starting with different amounts (5μg, 1μg, 200ng, 20ng, 2ng, 100pg) of human brain total RNA, the All-inOne miRNA qRT-PCR Detection Kit was used to detect the expression level of hsa-miR-124. The results
showed that linear amplification can be detected between 5μg ~ 100pg of total RNA.
R2＝0.997
E＝103.9％
Figure 3. The amplification curve and standard curve generated from different amount of human
brain total RNA as template, and using All-in-One miRNA qRT-PCR Detection Kit to detect hsa-miR124 expression level.
9
All-in-One™ miRNA qRT-PCR Detection Kit User Manual
VII. Trouble Shooting Guide
• Because the sequence of miRNA is short and some have a high
degree of homology, the primer design can sometimes be tricky. Thus
miRNA sequence
homology problems
one needs to fully consider the specificity problems when designing
the miRNA forward primers. Specifically for miRNAs that have a
single nucleotide difference only, the demand for specificity is higher
for designing and synthesizing primers, in addition to designing
reaction conditions.
• The fluorescence detection temperature may not be appropriate.
Adjust accordingly.
Confusion of
amplification curves
Abnormal melting
curves
• The set up position for samples may not be right. Adjust accordingly.
• Try to use 3.5% agarose gel electrophoresis to check the PCR
products. Check the purity of the primers using electrophoresis or use
PAGE-purified primers if the bands are diffused. One may also use
phenol/chloroform extraction and ethanol precipitation methods to
treat the primers before experiment.
• Signals in blank (No Template Control) sample
• There may be contamination or positive samples in the PCR reaction
system if the T
as the positive control. Eliminate sample application error first. If the
situation still persists, change PCR grade water, primers or use new
2×All-in-One
• If the T
control, the PCR reaction may have produced nonspecific
amplification such as primer-dimers. Please prepare PCR reaction
mix on ice and increase the temperature of fluorescence detection. If
the C
positive samples is more than 5 cycles, the PCR reaction system is
up to the standard. On the other hand, if the
aforementioned value, then redesign the primer or optimize the
reaction conditions.
of melting curves of blank control is lower than the positive
m
value of the negative control is >35 and the difference with the
t
of the melting curves of the blank control is the same
m
TM
q-PCR Mix.
value cannot reach the
Ct
• Double peaks and multiple peaks in melting curves of positive
control
• In the absence of other primers present in the reaction, double or
multiple peaks in the positive control means that the PCR reaction
produces nonspecific amplification fragments. Prepare the PCR
reaction mix on ice; optimize the PCR reaction conditions such as by
increasing the annealing temperature, decreasing the primer
10
All-in-One™ miRNA qRT-PCR Detection Kit User Manual
No signal (Ct) or Ct
value is too high
concentration or increasing the fluorescence detection temperature
(no more than the Tm value of the expected product). If this does not
work, optimize and redesign the forward primer.
• Check if there are PCR products to exclude the possibility of
instrument detector malfunction.
• Not enough PCR cycles. For good sensitivity, one should generally
set up more than 35 PCR cycles, but more than 45 cycles may result
in too much background signals.
• The amount of template may not be enough or the template may be
degraded. Use the highest concentration of diluted template samples
possible to set up PCR. At the same time, avoid freezing and thawing
samples repeatedly.
• Amplification efficiency is low and PCR reaction conditions are not
optimal. Redesign primers and optimize reaction conditions.
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All-in-One™ miRNA qRT-PCR Detection Kit User Manual
VIII. Limited Use License and Warranty
Limited Use License
Following terms and conditions apply to use of all OmicsLink™ ORF Expression Clones in all lentiviral vectors and Packaging Kit (the
Product). If the terms and conditions are not acceptable, the Product in its entirety must be returned to GeneCopoeia within 5 calendar days.
A limited End-User license is granted to the purchaser of the Product. The Product shall be used by the purchaser for internal research
purposes only. The Product is expressly not designed, intended, or warranted for use in humans or for therapeutic or diagnostic use. The
Product must not be resold, repackaged or modified for resale, or used to manufacture commercial products without prior written consent
from GeneCopoeia. This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic
research. Use of any part of the Product constitutes acceptance of the above terms.
Limited Warranty
GeneCopoeia warrants that the Product meets the specifications described in the accompanying Product Datasheet. If it is proven to the
satisfaction of GeneCopoeia that the Product fails to meet these specifications, GeneCopoeia will replace the Product. In the event a
replacement cannot be provided, GeneCopoeia will provide the purchaser with a refund. This limited warranty shall not extend to anyone
other than the original purchaser of the Product. Notice of nonconforming products must be made to GeneCopoeia within 30 days of receipt
of the Product. GeneCopoeia’s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price.
GeneCopoeia’s liability does not extend to any damages arising from use or improper use of the Product, or losses associated with the use
of additional materials or reagents. This limited warranty is the sole and exclusive warranty. GeneCopoeia does not provide any other
warranties of any kind, expressed or implied, including the merchantability or fitness of the Product for a particular purpose.
GeneCopoeia is committed to providing our customers with high-quality products. If you should have any questions or
concerns about any GeneCopoeia products, please contact us at 301-762-0888.
© 2009, GeneCopoeia, Inc.
GeneCopoeia, Inc.
9620 Medical Center Drive
Rockville, MD 20850
Tel: 301-762-0888 Fax: 301-762-3888
Email:
Web:
HUinquiry@genecopoeia.comU
HUwww.genecopoeia.comU
GeneCopoeia Products are for Research Use Only Copyright © 2009 GeneCopoeia, Inc.
Trademarks: GeneCopoeia™, OmicsLink™, All-in-One™, EndoFectin™, miTarget™, miExpress™ (GeneCopoeia Inc.); SYBR
iQ™5 (Bio-Rad); Trizol™, ABI
®
, ROX® (Invitrogen); NanoDrop™ (Thermo Scientific). UMAOM002
12
®
(Molecular Probes);
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