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RMQ-101, RMQ-101T
Instruction manual for QuantAccuracyTM, RT-RamDATM cDNA Synthesis Kit Dec. 2021

QuantAccuracy™,
RT-RamDA™ cDNA Synthesis Kit
RMQ-101 96 reactions
RMQ-101T 24 reactions

Store at minus (-) 20℃

Contents[1]

Introduction[2]

Components[3]

Protocol1

For Cell Samples
1-1. Cell lysis
1-2. Denaturation
1-3. Digestion of genomic DNA
1-4. cDNA synthesis and amplification (RT-RamDA™)

2

For RNA Samples
2-1. RNA dilution
2-2. Denaturation
2-3. cDNA synthesis and amplification (RT-RamDA™)[4]

Application data

[5]

Troubleshooting[6]

Related products

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnostic or clinical purposes. Please observe
general laboratory safety precautions while using this kit.
* RT-RamDA™ and RamDA-seq™ are trademarks of RIKEN, Institute of Physical and Chemical Research, JapanJAPANTOYOBO CO., LTD.
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RMQ-101, RMQ-101T[1]

Introduction
Description
The QuantAccuracy™, RT-RamDA™ cDNA Synthesis Kit (Code No. RMQ-101) is an
improved version of the RT-RamDA™ cDNA Synthesis Kit (Code No. RMD-201) for
preparing cDNA from single cells or trace amounts of RNA for real-time PCR analysis.
Using this kit, cDNA can be prepared from full-length total RNA, and gene expression
analysis can be performed with high sensitivity.
This kit uses reverse transcription with random displacement amplification (RTRamDA™) (see method in reference (1)). RT-RamDA™ is a novel cDNA amplification
method that utilizes the strand displacement activity of reverse transcriptase, which candetect not only poly(A) RNA but also non-poly(A) RNA with high sensitivity. For thisreason, the RT-RamDA™ method is characterized by its ability to detect more genes thanconventional technology.

Process Workflow
Features
1. cDNA can be prepared from a single cell or a small amount of input RNA
Between 1–500 cells or 10 pg–10 ng total RNA should be used.
2. Low-copy genes can be analyzed
By amplifying RNA to tens of times the amount of cDNA, low-copy genes can be analyzed
from samples containing small amounts of RNA.
3. Very accurate quantification
Amplification occurs randomly in RT-RamDATM, which can reduce amplification bias.
4. Minimize input samples
Because amplified cDNA can be obtained, valuable samples are saved.
5. Can be used with a variety of real-time PCR reagentsThis kit can be used in combination with a variety of real-time PCR reagents and iscompatible with both SYBR® Green I and TaqMan® assays.
THUNDERBIRD® qPCR Mix (Code No. QPS-101, QPS-201, QPX-201), and KOD
SYBR® qPCR Mix (Code No. QKD-201) can be used.

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RMQ-101, RMQ-101T[2]

Components
The kits include the following reagents that can be used for 96-well (RMQ-101) or 24-well(RMQ-101T) reactions. All reagents should be stored at -20℃.
Important: Do not store any of the solutions once mixed. All reagent cocktails should be
prepared shortly before use.

QuantAccuracy™, RT-RamDA™ cDNA Synthesis Kit (Cat. No. RMQ-101 and RMQ101T)①Lysis Buffer②Lysis Enhancer③RNase Inhibitor④RT-RamDA™ Buffer⑤gDNA Remover⑥RT-RamDA™ Enzyme Mix⑦RT-RamDA™ Primer Mix⑧Nuclease-free water

RMQ-101
480 μL108 μL
22 μL
160 μL
54 μL
60 μL
60 μL
960 μLRMQ-101T
120 μL
27 μL
6 μL
40 μL
14 μL
15 μL
15 μL
240 μL

Required materials not included in the kit
Thermocycler
Equipment and reagents for real-time PCRJAPANTOYOBO CO., LTD.
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RMQ-101, RMQ-101T[3]

ProtocolThe kit can be used with cells or purified total RNA. Start with Section 1A when using
FACS to obtain the cells, Section 1B when using non-FACS cells, and Section 1C whenusing purified total RNA. Thereafter, proceed with denaturation as outlined in Section 2.
SampleCells

Cell lysis or RNA dilution
Section 1-1. A (p. 1)
Section 1-1. B (p. 2)
Section 2-1. (p. 5)

FACS
Non-FACS

Purified RNA

Denaturation
Continue with Section
1-2. (p. 2)
Continue with Section
2-2. (p. 6)

1. When using cells
1-1. Cell lysis
A. When acquiring cells by FACS
(1) Prepare a cocktail of the cell lysis reagents in a single tube using the volumes listed in
the table below. The volumes used will need to be adjusted depending on the number ofreactions that you wish to perform.
Cell lysis cocktail
1 Reaction(μL)①Lysis Buffer②Lysis Enhancer③RNase Inhibitor⑧Nuclease-free water
Total

2
0.45
0.05
2.5
5

20 Reactions
(μL)*
44
9.9
1.1
55
110

100 Reactions
(μL)*
220
49.5
5.5
275
550

*An extra 10% volume was added here to account for pipetting error.

(2) Dispense 5 μL of cell lysis cocktail per well into a 96-well plate or 8-tube strip.
Dispense on ice, and immediately after dispensing, seal with a qPCR seal orthermocompression seal.
(3) Store the plate on ice or at 4℃ until cell sorting according to the FACS user manualand the parameters recommended by the manufacturer.
(4) After sorting, seal the plate or tubes and then centrifuge briefly to collect the solution
at the bottom of the wells or tubes.
(5) Proceed immediately to the next step or store the samples at -80℃.

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RMQ-101, RMQ-101T
B. Cell acquisition using methods other than FACS
When obtaining the cells by manual picking, the volume of the cell sample should be 1.5
μL or less.
(1) Prepare the reagents necessary for cell lysis in one tube per reaction using the volumes
listed in the table below, and then add the cell samples.
If the volume of the cell sample is less than 1.5 μL, adjust the amount of water added when
preparing the cell lysis cocktail.
1 Reaction (μL)
①Lysis Buffer②Lysis Enhancer③RNase Inhibitor
Sample⑧Nuclease-free water
Total

2
0.45
0.05
~1.5
X (adjust as needed based on
sample volume)5

For example, if your cell sample volume is 1 μL, prepare the cell lysis cocktail using thevolumes listed in the table below, and dispense 4 μL of the cell lysis cocktail per well into
a 96-well plate or 8-strip tube. Then, add 1 μL of your cell samples.
Cell lysis cocktail
1 Reaction (μL)
①Lysis Buffer②Lysis Enhancer③RNase Inhibitor⑧Nuclease-free water
Total

2
0.45
0.05
1.5
4

20 Reactions(μL) *
44
9.9
1.1
33
88

100 Reactions(μL) *
220
49.5
5.5
165
440

*An extra 10% volume was added here to account for pipetting error.
(2) Seal the plate or tubes and then centrifuge briefly to collect the solution at the bottomof the wells or tubes.
(3) Proceed immediately to the next step or store the samples at -80℃.

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RMQ-101, RMQ-101T
1-2. Denaturation(1) Centrifuge the sample plate or tubes at 4℃ and incubate them according to the tablebelow.
Note: When using frozen samples, first thaw the samples at 4℃ and then briefly centrifuge
them before proceeding to heat denaturation.
Step
Denature
Hold

Temperature
75℃
4℃

Time
1.5 min∞1-3. Digestion of genomic DNA(1) Prepare a cocktail of the reagents necessary for genomic DNA removal using thevolumes listed in the table below. The volumes used will need to be adjusted depending on
the number of reactions that you wish to perform.
DNA removal cocktail
1 Reaction(μL)④RT-RamDA™ Buffer⑤gDNA Remover⑧Nuclease-free water
Total

0.2
0.45
1.35
2

20 Reactions
(μL)*
4.4
9.9
29.7
44

100 Reactions
(μL)*
22
49.5
148.5
220

*An extra 10% volume was added here to account for pipetting error.
(2) Add 2 μL of the genomic DNA removal cocktail per well to the 96-well plate or 8-tube
strip containing the heat-denatured samples from the previous step. Centrifuge briefly, thenincubate at the following temperatures:
Step

Temperature

Genomic DNA digestion
Hold

30℃
4℃

Time
5 min
∞

(3) Proceed immediately to the next step after gDNA digestion has occurred.

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RMQ-101, RMQ-101T
1-4. cDNA synthesis and amplification reaction (RT-RamDA™ reaction)(1) Prepare a cocktail of the necessary reagents for the RT-RamDA™ reaction in a single
tube using the volumes listed in the table below. The volumes used will need to be adjusted
depending on the number of reactions that you wish to perform.
RT-RamDA™ cocktail
1 Reaction(μL)④RT-RamDA™ Buffer⑥RT-RamDA™ Enzyme Mix⑦RT-RamDA™ Primer Mix
Total

1
0.5
0.5
2

20 Reactions
(μL)*
22
11
11
44

100 Reactions
(μL)*
110
55
55
220

*An extra 10% volume was added here to account for pipetting error.

(2) Add 2 μL of RT-RamDA™ cocktail solution per well to the 96-well plate or 8-tube
strips in which the genomic DNA removal reactions were performed. After brief
centrifugation, incubate the plate or tubes at the following temperatures:
Step
Priming 1

Temperature
25℃

Time
10 minPriming 2

30℃

10 min

Reverse transcription andamplification

37℃

30 min50℃

5 min

Inactivation

98℃

5 min

Hold

4℃

∞

(3) Proceed immediately to the next step after the RT-RamDA™ reaction, or store the plateor tubes at -20℃ to -30℃.
(4) When performing real-time PCR, add the RT-RamDA™ reaction solution directly or
diluted to the reaction solution as a template.
In real-time PCR, the template amount of the RT-RamDA™ reaction solution should be
limited to less than 10% of the total volume. For example, if the total volume is 20 μL for
real-time PCR, up to 2 μL of RT-RamDA™ reaction solution can be applied. The additionof a large amount of RT-RamDA™ reduces the efficiency of PCR, and accuratequantification may no longer be possible.

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RMQ-101, RMQ-101T
2. When using purified total RNA
2-1. RNA dilution(1) Prepare the reagents necessary for RNA dilution in one tube per reaction using thevolumes listed in the table below and then add the cell samples.
If the volume of the cell sample is less than 2.5 μL, adjust the amount of water.
1 Reaction (μL)
①Lysis Buffer②Lysis Enhancer③RNase Inhibitor
Sample⑧Nuclease-free water
Total

2
0.45
0.05
~2.5
X (adjust as needed based on
sample volume)5

For example, if the RNA sample volume is 2 μL, prepare the RNA dilution cocktail using
the volumes listed in the table below, and dispense 3 μL of the RNA dilution cocktail per
well into a 96-well plate or 8-strip tube. Then, add 2 μL of the RNA samples.
RNA dilution cocktail
1 Reaction (μL)
①Lysis Buffer②Lysis Enhancer③RNase Inhibitor⑧Nuclease-free
water
Total

20 Reactions(μL) *

2
0.45
0.05
0.5

44
9.9
1.1
11

100 Reactions(μL) *
220
49.5
5.5
55

3

66

330

*An extra 10% volume was added here to account for pipetting error.
(2) After dispensing, seal the tubes and centrifuge briefly to collect the solutions at thebottom of the wells or tubes.
(3) Proceed immediately to the next step or store the samples at -80℃.

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RMQ-101, RMQ-101T
2-2. Denaturation(1) Centrifuge the sample plate or tubes at 4℃ and incubate as described in the table below.
Note: When using frozen samples, first thaw the samples at 4℃, and then proceed to heat
denaturation after brief centrifugation.
Step
Denature
Hold

Temperature
75℃
4℃

Time
1.5 min∞2-3. cDNA synthesis and amplification reaction (RT-RamDA™ reaction)(1) Prepare a cocktail of reagents necessary for the RT-RamDA™ reaction using thevolumes listed in the table below. The volumes used will need to be adjusted depending on
the number of reactions that you wish to perform.
RT-RamDA™ cocktail④RT-RamDA™ Buffer⑤gDNA Remover⑥RT-RamDA™ Enzyme Mix⑦RT-RamDA™ Enzyme Mix⑧Nuclease-free water
Total

1 Reaction
(μL)
1.2
0.45
0.5
0.5
1.35
4

20 Reactions
(μL)*
26.4
9.9
11
11
29.7
88

100 Reactions
(μL)*
132
49.5
55
55
148.5
440

*An extra 10% volume was added here to account for pipetting error.
(2) Add 4 μL of the RT-RamDA™ cocktail per well to the 96-well plate or 8-tube strip
containing the heat-denatured samples from the previous step. Centrifuge briefly, and thenincubate at the following temperatures:
Step
Priming 1

Temperature
25℃

Time
10 minPriming 2

30℃

10 min

Reverse transcription andamplification

37℃

30 min

Reverse transcription 2

50℃

5 min

Inactivation

98℃

5 min

Hold

4℃

∞

(3) Proceed immediately to the next step after the RT-RamDA™ reaction or store the plateor tubes at -20℃ to -30℃.

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RMQ-101, RMQ-101T(4) When performing real-time PCR, add the RT-RamDA™ reaction solution directly or
diluted to the reaction solution as a template.
In real-time PCR, the template amount of the RT-RamDA™ reaction solution should be
limited to less than 10% of the total volume. For example, if the total volume is 20 μL in
real-time PCR, up to 2 μL of RT-RamDA™ reaction solution can be applied.
The addition of a large amount of RT-RamDA™ reduces the reaction efficiency of the
PCR, and accurate quantification may no longer be possible.

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RMQ-101, RMQ-101T[4]

Application data

Application 1. Comparison of the amount of cDNA between the QuantAccuracy™
and conventional reverse transcription kits
<Method>
cDNA synthesis was performed using 1 ng of total RNA extracted from HeLa S3 cells tocompare the QuantAccuracy™, RT-RamDA™ cDNA Synthesis Kit (QuantAccuracy,
Code No. RMQ-101) with a conventional reverse transcription kit from competitor A.
Real-time PCR analysis was then performed on two types of long non-coding RNA genes(NEAT1 and MALAT1) and seven types of housekeeping genes (ACTB, GAPDH, ATP5F1A,
YWHAZ, PPIA, B2M, RPS18, and HPRT1) using THUNDERBIRD® SYBR® qPCR Mix(Code No. QPS-201). Each cDNA was used as a template in a 20 μL reaction volume. The
amount of cDNA obtained using the QuantAccuracy™, RT-RamDA™ cDNA Synthesis
Kit was calculated from the Ct value, and the value obtained by dividing by the amount ofcDNA in the conventional reverse transcription reaction was calculated as theamplification rate.
<Results>
There was a significantly high correlation between the Ct values of the QuantAccuracy™
and reverse transcription kits (Figure 1). In addition, the average ΔCt values of the genes
between the QuantAccuracy™ and reverse transcription kits were > 5 (Figure 2). Thus, byusing the QuantAccuracy™, RT-RamDA™ cDNA Synthesis Kit, it is possible to obtain
30 times or more cDNA than that obtained using the conventional reverse transcription
reaction reagent.

Figure 1. Correlation between QuantAccuracy™ (y-axis) and a conventional reverse
transcription kit (x-axis, Company A).

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RMQ-101, RMQ-101T

Figure 2. ΔCt values between QuantAccuracy™ and a conventional reverse transcription
kit

Application 2. Analysis using a human FFPE tissue<Method>
cDNA synthesis was performed using 100 pg or 1 ng RNA extracted from human FFPE
tissue to compare the QuantAccuracy™, RT-RamDA™ cDNA Synthesis Kit (Code No.
RMQ-101) with other cDNA amplification kits from competitors B and C, and aconventional reverse transcription kit from competitor D. As in Application 1, real-time
PCR analysis was performed on two types of long non-coding RNA genes and seven types
of housekeeping genes using THUNDERBIRD® SYBR® qPCR Mix (Code No. QPS-201)with each cDNA used as a template.
The amount of cDNA in the QuantAccuracy™, RT-RamDA™ cDNA Synthesis Kit wascalculated from the Ct value, and the value obtained by dividing by the amount of cDNAin the conventional reverse transcription reaction was calculated as the amplification rate.
<Results>
First, compared to the other products, QuantAccuracy™ exhibited the highest detection
rate for all genes (Table 1).
In addition, among these products, only QuantAccuracy™ yielded parallel line graphs
connecting Ct values of the genes in the study with 100 pg and 1 ng FFPE RNA, indicatingthat constant amplification occurred irrespective of the type of genes (Figure 3). The
average ΔCt value in the study using QuantAccuracy™ was calculated to be approximately
3.40, indicating that the 10-fold difference between FFPE RNAs was 23.40 ≈ 10.6 times
difference (Figure 3). Thus, the QuantAccuracy™, RT-RamDA™ cDNA Synthesis Kit
allows highly accurate analysis despite the use of FFPE tissues n this study.

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RMQ-101, RMQ-101T
Table 1. Detection rates of the genes

Figure 3. Line graphs connecting the Ct values of the genes
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RMQ-101, RMQ-101T[ 5 ] Troubleshooting

Symptom
No signal ordelayed detection
in real-time PCR

[6]

Cause

Solution

RNA is degraded· Check whether RNA has degraded.
· Check whether RNase has contaminated the tips or tubes used.
· Perform all cell lysis and RNA dilution steps on ice.

Excess reverse
transcription reaction
solution added

If excess amount is added, PCR may be inhibited. The amount ofreverse transcription reaction solution added to the real-time PCR
reaction solution should be 10% or less of the amount of PCR solution.

Related products

Product name
GenNext™ RamDA-seq ™ Single Cell Kit

No. of reactions
96
24
96
24
500
500
500
500

cDNA prep kit for NGS

RT-RamDA™ cDNA Synthesis Kit
THUNDERBIRD® Probe qPCR Mix
THUNDERBIRD® SYBR® qPCR Mix
THUNDERBIRD® Next SYBR® qPCR Mix
KOD SYBR® qPCR Mix

Catalog No.
RMD-101
RMD-101T
RMD-201
RMD-201T
QPS-101
QPS-201
QPX-201
QKD-201

Reference 1
Tetsutaro Hayashi*, Haruka Ozaki*, Yohei Sasagawa, Mana
Umeda, Hiroki Danno and Itoshi Nikaido. Single-cell full-length
total RNA sequencing uncovers dynamics of recursive splicing andenhancer RNAs. Nature Communications. 2018.

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