Safety information - Eurogentec

MANUAL FOR 

MiRmaid miRNA PRECURSORS qRT-PCR PRIMER SET (HUMAN) 

+ qPCR MASTERMIX PLUS FOR SYBR ® GREEN I 

REFERENCE NUMBER: RT-PRMI-01

2 

Table of contents 

Intended use 3 

Shipping and storage conditions 3 

Safety information 3 

Background Information 4 

Kit contents 6 

MiRmaid miRNA precursors qRT-PCR primer set components 7 

Additional required material 7 

Standard protocol 8 

1. Reagent handling 8 

2. RNA preparation 8 

3. DNase and Reverse Transcription Reactions 9 

4. Quantitative qPCR Reaction 10 

5. How to set up a good assay 12 

Typical results 14 

Troubleshooting guide 15 

Homology between human pre-miRNA primers and rodent sequences 19 

Guidelines for successful RT-qPCR 20 

Ordering information 21 

Related products 23 

Appendix 1: Layout of MiRmaid qPCR primer plate #1 24 

Appendix 2: Layout of MiRmaid qPCR primer plate #2 24 

Appendix 3: qPCR MasterMixes compatibility with Real-Time thermocyclers 25 

Trademarks and disclaimer 26 

Manual for MiRmaid miRNA precursor qRT-PCR Primer Set I www.eurogentec.com I info@eurogentec.com

Intended use 

The Eurogentec MiRmaid miRNA precursors qRT-PCR primer set – designed by DNA 

Vision - was developed and optimized for the amplification of the human precursor 

microRNAs (pre-miRNAs) by performing a reverse transcription (RT) followed by a 

Real-Time qPCR using SYBR ® Green I assay. 

This product is sold for research use only. The kit is not intended for use in diagnostic 

or therapeutic procedures. Do not use internally or externally in humans or animals. 

All miRNAs that can be amplified with the MiRmaid miRNA precursors qRT-PCR primer set are 

listed in appendix 1 and 2 (as miRNA names evolve quickly along time you will find a conversion 

table on our website giving the new miRNA names corresponding to our miRNA MiRmaid names). 

Reference center: Dr. Ogier D. - INSERM U773 - Centre de recherche Bichat Beaujon - CRB3 - Paris - France 

Shipping and storage conditions 

The MiRmaid miRNA precursors qRT-PCR primer set kit is shipped on dry ice. 

Upon receipt, the MiRmaid RT primer MasterMIX, the 2 MiRmaid qPCR primer plates, 

the qPCR MasterMix Plus for SYBR ® Green I and the extra tube 50 mM MgCl 2 should 

be stored at -20 °C (-18 °C to –24 °C) in a constant temperature freezer to maintain 

stability. Repeated freeze-thaw cycles (more than 5) should be avoided. 

When stored under these conditions the reagents are stable for at least 1 year. 

The qPCR MasterMix Plus for SYBR ® Green I should be protected from light whenever possible. 

Safety information 

This product and its components should be handled only by persons trained in 

laboratory techniques and used in accordance with the principles of good laboratory 

practice. All chemicals should be considered potentially hazardous. Therefore, when 

handling chemical reagents, it is advisable that suitable protective clothing, gloves and 

eye/face protection are worn. Care should be taken to avoid contact with skin or eyes. 

In case of contact, wash immediately with water and seek medical advice. 

DMSO, in which SYBR ® Green I is dissolved, may cause eye, skin, and respiratory tract 

irritation. It is readily absorbed through the skin. DMSO is a combustible liquid and 

vapor that should be kept away from heat and flame. The product contains material, 

Manual for MiRmaid miRNA precursor qRT-PCR Primer Set I www.eurogentec.com I 

info@eurogentec.com 3

4 

which may cause liver and blood damage. We recommend wearing a lab coat, 

disposable gloves, and protective goggles when working with these chemicals. 

For more information about safety and handling of reagents please consult the 

Material Safety Data Sheets (MSDSs) of this kit and its components. These are 

available as PDFs on our website at www.eurogentec.com/code/en/msds.asp. 

Background information 

Figure 1 : miRNA Biogenesis and siRNA Pathway 

MicroRNAs (miRNA) represent an abundant class of endogenous non-coding RNA 

molecules that regulate fundamental processes such as cell proliferation, cell 

differentiation or apoptosis through modulation of gene expression (1 and 2). 


miRNA genes are usually transcribed by RNA Polymerase II into capped, 

polyadenylated and spliced transcripts (cf figure 1). miRNA-containing sequences 

from one or more hairpin loop structures that are called primary miRNA structure 

(pri-miRNA). The RNase III Drosha-DGCR8 nuclear complex cleaves the base of the 

hairpin to form a precursor molecule called pre-miRNA (3-5). Most pre-miRNAs are 

comprised between 70-120 nt with 1-4 nt 3' overhangs, and form a 25–30 bp hairpin 

loop structure. The pre-miRNA molecule is then actively transported out of the 

nucleus into the cytoplasm by Exportin 5, a carrier protein. The Dicer enzyme (a key 

enzyme of the siRNA pathway) then cuts 19-25 nucleotides from the base of the 

hairpin (approximately 19 bp from Drosha's cut site) to release a double-stranded RNA 

molecule with 1–4 nt 3' overhangs at either end (6,7). Only one strand of the duplex 

(called the mature miRNA) is incorporated into the RISC (RNA-Induced Silencing 

Complex) or miRNP complex, a protein complex that, when bound to a miRNA, is 

responsible for the post-transcriptional regulation of the corresponding target protein 

by an unknown mechanism. 

In the RNA interference (RNAi) phenomenon, the Dicer also processes the synthetic 

small interfering RNA (siRNA) from longer precursors. The antisense strand of the 

siRNA serves as a template for the RISC to recognize and cleave a complementary 

messenger (cf figure 1, siRNA pathway). 

The mature miRNAs have been shown to reduce steady state protein levels for the 

targeted gene(s) by binding on its 3’UTR with or without impacting the corresponding 

levels of mRNA. The mechanisms by which miRNAs reduce protein levels are not fully 

understood. These post-transcriptional events, would take place in the dynamic 

cytoplasmic structures, called P-body or GW body (8,9-11). Moreover, a single miRNA 

can bind to and regulate many different mRNA targets or can bind at several positions 

on a same UTR. Conversely, several different miRNAs can bind to and cooperatively 

control a single mRNA. 

Several studies indicate that miRNAs may regulate the translation as much as 30 % of 

all genes in the genome. miRNAs have already been found to involve into several types 

of cancers, cell differentiation, proliferation and viral infection. miRNA expression 

varies from tissue to tissue and looks also highly regulated during the development in 

a temporal way. 

Interestingly, a recent study performed at the MIT on different types of cancer has 

shown that miRNAs signatures are more informative than classical mRNAs ones. They 

observed so far an increase of the abundance of the miRNAs matures in cancer 

compare to the normal situation without the detection of a substantial decrease in the 

mRNAs encoding components of the miRNAs processing machinery (Dicer, Drosha, 

Argonaute 2 or DGCR8). This observation suggests either the existence of other 



6 

mechanisms of regulation or it is the expression of the miRNAs, which is affected, or 

therefore the analysis of the expression of the pre-miRNAs become highly relevant (12). 

Reference 

1. Bartel, DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281-97 

2. Lai, EC. microRNAs: runts of the genome assert themselves. Curr Biol 2003; 13: R925-36 

3. Hutvagner, G, McLachlan, J, Pasquinelli, AE, Balint, E, Tuschl, T and Zamore, PD. A cellular function for the RNAinterference 

enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 2001; 293: 834-8 

4. Lee, Y, Ahn, C, Han, J, Choi, H, Kim, J, Yim, J, Lee, J, Provost, P, Radmark, O, Kim, S and Kim, VN. The nuclear RNase 

III Drosha initiates microRNA processing. Nature 2003; 425: 415-9 

5. Lee, YS, Nakahara, K, Pham, JW, Kim, K, He, Z, Sontheimer, EJ and Carthew, RW. Distinct roles for Drosophila Dicer- 

1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 2004; 117: 69-81 

6. Khvorova, A, Reynolds, A and Jayasena, SD. Functional siRNAs and miRNAs exhibit strand bias. Cell 2003; 115: 209-16 

7. Schwarz, DS, Hutvagner, G, Du, T, Xu, Z, Aronin, N and Zamore, PD. Asymmetry in the assembly of the RNAi enzyme 

complex. Cell 2003; 115: 199-208 

8. Liu, J, Valencia-Sanchez, MA, Hannon, GJ and Parker, R. MicroRNA-dependent localization of targeted mRNAs to 

mammalian P-bodies. Nat Cell Biol 2005; 7: 719-23 

9. Pauley, KM, Eystathioy, T, Jakymiw, A, Hamel, JC, Fritzler, MJ and Chan, EK. Formation of GW bodies is a consequence 

of microRNA genesis. EMBO Rep 2006; 7: 904-10 

10. Rehwinkel, J, Behm-Ansmant, I, Gatfield, D and Izaurralde, E. A crucial role for GW182 and the DCP1:DCP2 

decapping complex in miRNA-mediated gene silencing. Rna 2005; 11: 1640-7 

11. Bruno, I and Wilkinson, MF. P-bodies react to stress and nonsense. Cell 2006; 125: 1036-8 

12. Lu, J, Getz, G, Miska, EA, Alvarez-Saavedra, E, Lamb, J, Peck, D, Sweet-Cordero, A, Ebert, BL, Mak, RH, 

Ferrando, AA, Downing, JR, Jacks, T, Horvitz, HR and Golub, TR. MicroRNA expression profiles classify 

human cancers. Nature 2005; 435: 834-8 

Kit contents 

The Eurogentec MiRmaid miRNA precursors qRT-PCR primer set (reference 

RT-SYMI-025) contains sufficient MiRmaid RT primer MasterMIX to perform 50 RT 

(Reverse Transcriptase) reactions, which is enough for 25 different samples. The amount 

of cDNA produced after 1 RT reaction should allow performing 96 qPCR reactions. 

The qPCR MasterMix Plus for SYBR ® Green I is a 2X concentrated, ready-to use reaction 

buffer containing dNTP/dUTP mix, HotGoldStar, UNG, MgCl 2, SYBR ® Green I, stabilizers 

and ROX passive reference. An extra tube of 50 mM MgCl 2 is provided. The qPCR 

MasterMix Plus for SYBR ® Green I contains enough reagents for performing up to 600- 

25 μl reactions. 

If for the Real-Time thermocycler that is used another qPCR MasterMix is required, please refer to our 

Real-Time qPCR MasterMix compatibility table with each Real-Time thermocyclers in appendix 3. 

The 2 MiRmaid qPCR primer plates contain 112 μl of the specific miRNA Reverse and 

Forward primers in each tube. These 170 pre-miRNA F and R primer mixes and the 

4 positive control mixes are sufficient to perform at least 25 Real-Time qPCR reactions. 

If the qPCR analysis should be performed in duplicates please order a second qPCR MasterMix 

Plus for SYBR ® Green I separately (reference # RT-SN2X-03+). 


MiRmaid miRNA precursors qRT-PCR primer set 

components 

2x reaction buffer (amber bottle - blue cap)* 7.5 ml 

• One bottle of reaction buffer containing dNTP/dUTP mix, HotGoldStar, UNG, MgCl 2 (5 mM 

final concentration), SYBR ® Green I, stabilizers and passive reference 

• Sufficient to perform 600-25 μl reactions. 

• Store at –20 °C 

(* ) The SYBR ® Green is light sensitive and should be kept away from light as much as possible 

50 mM MgCl 2 (plain cap) 1.5 ml 

• One tube of 50 mM MgCl 2 

• Store at -20 °C 

MiRmaid RT primer MasterMIX (red cap) 765 μl 

• One tube of specific miRNA RT primers at 10 μM each 

• Sufficient to perform 50 RT reactions (25 samples) 


2 MiRmaid qPCR primer plates 112 μl 

• Two plates with 170 tubes containing 112 μl of specific miRNA Forward and Reverse qPCR 

primers at 2.5 μM. 4 positive controls are included in each plate (112 μl of specific Forward 

and Reverse primers at 2.5 μM) 

• Sufficient to perform at least 25 qPCR using 4 μl. 


Layout of the 2 plates can be found in the cover of each plate and in appendix 1 and 2 of this manual. 

Additional required material 

• Real-Time Thermocycler 

• Deoxyribonuclease I, Amplification grade 

• SuperScript III Reverse Transcriptase 

• dNTP 10 mM each (Reference NU-0020-10) 

• QIAquick Nucleotide Removal Kit, Qiagen cat 28304 

• Sterile, RNase-free 1.5 ml microtubes 

• Sterile-barrier, RNase-free pipette tips 

• RNase-free water 

• EDTA (10mM, pH 8.0) 

• DTT (0.1 M) - provided with the SuperScript III 

• PCR-tubes 



8 

Standard protocol 

1. Reagent handling 

• Thaw all required reagents completely and put them on ice. 

• Mix all reagents well by inversion and spin them down prior to pipetting. 

2. RNA preparation 

The quality of the RNA is essential to the overall success of the all data from MiRmaid 

miRNA precursors qRT-PCR primer set. 

• Clean your work area before starting with RNAZap product or equivalent. 

• Wear clean lab coat and gloves at any time (and change them whenever you suspect 

that they are contaminated). 

• Use RNase free tips and tubes. 

Tips 

The pre-miRNAs look highly sensitive to RNA degradation, therefore the quality of the 

RNA should be checked before and after DNase treatment on an Agilent Bioanalyser 

2100 if possible (or at least on agarose gel figure 2). A ratio 260/280 of 1.8 - 2.0 as well 

as a perfect electropherogram are crucial to guarantee highly specific amplification. 

Moreover, because of the small size of the pre-miRNAs, it is recommended not using 

any total RNA extraction protocol based on exclusion column, as with this method all 

small RNAs would be lost. 

We recommend working on total 

RNA extracted with Trizol or RNABle 

RNA Extraction procedure. The 

figure 3 shows the analysis of total 

RNA after RNABle RNA Extraction, 

data obtained with the Agilent 

Bioanalyser 2100. 

Figure 2: Results on agarose gel 2 % 

TThhee ttoottaall RRNNAA iiss eexxttrraacctteedd wwiitthh tthhee RRNNAABBllee 

pprroocceedduurree.. 

A smear around 150 bp shows the presence of 

small RNAs after DNase treatment. 

1 2 3 4 

Before After 

DNase treatment 

Manual for MiRmaid miRNA precursor qRT-PCR Primer Set I www.eurogentec.com I info@eurogentec.com 

DNA ladder 

4000 bp 

800 bp 

350 bp 

150 bp

5S 

18S 

3. DNase and Reverse Transcription Reactions 

DNase treatment 

28S 

The first step after total RNA extraction is a treatment with a Deoxyribonuclease 

enzyme (DNase). This step is indispensable for removing DNA contamination from 

RNA samples prior to RT. 

1. Take 1 μg of total RNA (this quantity is enough for 96 qPCR reactions). 

2. Prepare a reaction mix by combining the following component in order as detailed 

below, to perform the DNase treatment. 

COMPONENT VOLUME 

Total RNA 1 μg (quantity is enough for 96 reactions) 

Deoxyribonuclease I, Amplification grade 1 μl 

DNase Buffer 1 μl 

RNase free water Volume is 10.5 μl minus all other component 

value 

Total mix 10.5 μl 

RT minus control: We recommend preparing 2 tubes in parallel with this mix preparation. One tube will be 

used as RT minus control in which no reverse transcription takes place in order to check whether the RNA 

preparation contains genomic DNA after the DNase treatment. 

3. Incubate 15 minutes at room temperature. 

4. Add 1 μl of EDTA (10 mM, pH8) to stop the DNase reaction. 

RT minus control: In the tube that will be used as no reverse transcriptase control, add 37.5 μl of RNase free 

water. Save it at -20 °C and use it directly in the step 15. 


Figure 3 : Result from Agilent Bioanalyser 

2100. 

TThhee ttoottaall RRNNAA iiss eexxttrraacctteedd wwiitthh tthhee RRNNAABBllee 

pprroocceedduurree.. 

Presence of the peak corresponding to the 

positive control 5S shows that small RNAs 

have been correctly extracted. 


10 

Reverse Transcriptase reaction 

5. Thaw the MiRmaid RT primer MasterMIX (red cap) completely and put it on ice. 

Vortex briefly and make a short spin by centrifugation. 

6. Add 15 μl MiRmaid RT primer MasterMIX to the RNA/DNase treated tube (from step 4). 

7. Incubate 5 minutes at 80 °C. 


9. Incubate 10 minutes at room temperature. 

10. Prepare a RT reaction mix by combining the following components, in order, as 

detailed below. 

COMPONENT VOLUME 

5x first strand buffer 9.6 μl 

DTT 4.8 μl 

dNTP (10 mM) 1.2 μl 

RNase free water 5.9 μl 

Total mix 21.5 μl 

11. Add the RT Reaction mix to RT primer MasterMIX + RNA tube (from step 6). 


13. Add 1.6 μl of Superscript III. 

14. Incubate for 60 minutes at 50 °C. 

15. Purify the product with the QIAquick Nucleotide Removal Kit and elute with 40 μl 

of EB buffer. 

16. Add EB buffer (+/- 285 μl) to the eluate so that the final volume of cDNA is 325 μl. 

4. Quantitative qPCR reaction 

1. Prepare a qPCR reaction mix by combining the following components in order as 

detailed below. It is recommended preparing a volume of reaction mix 10 % to 15 % 

greater than required for the total number of reactions (please find an example for 

96 reactions also in the table below). 


COMPONENT VOLUME PER WELL VOLUME FOR 96 REACTIONS 


+ 12 % (= 107.5 REACTIONS) 

2x reaction buffer 12.5 μl 1342 μl 

cDNA (from step 16) 3.0 μl 324 μl 

PCR Grade water 5.5 μl 594 μl 

Total MIX 21 μl 2260 μl 

2. Thaw the MiRmaid miRNA qPCR primer tubes necessary to perform the assay 

(please find the layout of the primer sets under the cover of each plate or in the 

appendix 1 and 2 of this manual). 

3. Make sure that the qPCR reaction mix is thoroughly mixed (do not vortex). Per well, 

aliquot 21 μl of this qPCR reaction mix and 4 μl of each MiRmaid qPCR primers. 

Final volume is 25 μl per well. Make sure that no bubbles are present in the tubes. 

Positive controls: It is recommended to perform positive controls using the 4 controls provided in each 

plate ( U6, 5S, HMBS and TBP). 

No-template controls: A negative control containing no cDNA template should also always be included. 

Do not forget to run the RT minus control. 

4. Centrifuge briefly to avoid bubbles. 

5a. Program the Real-Time thermocycler using the following cycling conditions: 

HotGoldStar activation 10 min 95 °C 

40 Cycles 15 sec 95 °C 

1 min 60 °C 

Data collection should be done at 60 °C. 

Perform a meltcuve to ensure the specificity of the product amplified. 

Dissociation Curve 15 sec 95 °C 

15 sec 60 °C 

(Ramping time: 19’59’’ with fluorescence reading) 

15 sec 95 °C 

5b. Program the Real-Time thermocycler using the following cycling conditions when 

using Uracil-N-Glycosylase: 

UNG step 2 min 50 °C 

HotGoldStar activation / UNG inactivation 10 min 95 °C 


12 

40 Cycles 15 sec 95 °C 

1 min 60 °C 

Data collection should be done at 60°C. 

Perform a meltcuve to ensure the specificity of the product amplified. 

Dissociation Curve 15 sec 95 °C 

15 sec 60 °C 

(Ramping time: 19’59’’ with fluorescence reading) 

15 sec 95 °C 

5. How to set up a good assay 

RNA quality 

The quality of the RNA is essential to the overall success of the all data from MiRmaid 

miRNA precursors qRT-PCR primer set. Clean laboratory practice is important to 

avoid degradation of the RNA (please find more information in paragraph 2 of the 

standard protocol). As the pre-miRNAs look highly sensitive to RNA degradation it is 

very important to check the quality of RNA before and after DNase treatment on an 

Agilent Bioanalyser 2100 if possible or at least on an agarose gel (please find more 

information p.8). Also, it is important to ensure that small RNAs have been well and 

correctly extracted using the 5S and U6 positive controls (please find more 

information concerning positive controls below). 

No-Reverse Transcriptase control 

As indicated in step 2 of the DNase and Reverse Tanscription Reactions paragraph, it 

is recommended performing a No-Reverse Transcriptase control. 

This control assumes the total digestion of the genomic DNA, which could interfere 

with the cDNA amplification. This control includes all of the qPCR reagents with 

primer controls and the RNA treated by DNase without the reverse transcription step. 

If a product is amplified, it indicates that there is still some genomic DNA 

contamination. 


Positive controls 

It is highly recommended including positive controls in the assay. This is why, 4 

different controls have been included in the 2 MiRmaid qPCR primer plates. U6 and 

5S are small size RNAs comparable to the size of precursor miRNAs. We recommend 

checking the presence of 5S before and after DNase treatment to ensure that the 

small size RNAs have been correctly extracted. By amplifying the U6 and 5S positive 

controls by qPCR you will be able to check that the qPCR reaction have been 

performed correctly and that any small RNAs reverse transcribed to cDNA have been 

amplified correctly. 

Precursor miRNAs are very low expressed RNAs, however 6U and 5S positive controls 

are highly expressed RNAs, this is why 2 others qPCR low expressed positive controls, 

HMBS and TBP, have been added. These 2 positive controls are coming out at Ct 

values that are more or less comparable to the Ct values expected when amplifying 

precursor miRNAs. 

No-template control 

It is recommended for each sample tested performing a negative control. 

The no-template control includes all the qPCR reagents except the cDNA / DNA 

template. If a product is amplified, it indicates that one or more of the qPCR reagents 

are contaminated with DNA. 

Dissociation curve 

The specificity of the amplification must be checked by the dissociation curve to 

assume the validation of the Cts. Each amplification is associated to a specific 

prominent peak corresponding to the target pre-miRNA amplification product. 

Standard curve 

A stantard curve should always be used to check the performance and efficiency of 

the qPCR. The standard curve should cover the complete range of expected 

expression levels (in general 5 to 6 logs of magnitude). The equation that represents 

this curve can be used to accurately identify fold-variation between experimental 

samples. 

All the MiRmaid qPCR primers have been designed to perform comparable qPCR 

efficiency when using Eurogentec qPCR MasterMix Plus for SYBR ® Green I. Thus, this 

also allows the quantification with the classical ΔΔCt method. 

4 positive internal controls have been included in the MiRmaid qPCR primer plates: 

U6, 5S, HMBS and TBP, which can be used as “housekeeping genes” and therefore 

also could be used to normalize the results. 



14 

Typical results 

An assay performed with MiRmaid miRNA precursor qRT-PCR primer set is fast, 

sensitive and optimized to amplify the miRNA precursors. The following experimental 

data were obtained with 1 μg of total RNA extracted from lung cells. The assays were 

performed according to the procedure described above. 

Figure 4: 

Amplification plots using the qPCR MasterMix Plus 

for SYBR ® Green I (RT-SN2X-03+) and some miRNA 

primers from MiRmaid qPCR primer plates on an 

ABI Prism ® 7900 HT 

Figure 5: 

Melting-curve analysis of six miRNA precursor 

RT-qPCR products on the ABI Prism ® 7900 HT 


Troubleshooting guide 

This troubleshooting guide takes you through the most common difficulties you can meet 

when performing a SYBR ® assay with the MiRmaid miRNA precursors qRT-PCR primer 

set. Our scientific support is happy to help you if you might have any further questions 

(info@eurogentec.com / info.uk@eurogentec.com / info.usa@eurogentec.com). 

No product from experimental sample is amplified 

and positive control does not work as expected 

Forgot one of the component 

Check again if all the steps have been performed and verify the thermal cycler 

conditions. 

RNA has been damaged or degraded 

Replace RNA if necessary. 

Quality of RNA 

If RNA is not of good quality the DNase treatment can have a negative effect on the 

starting material > replace RNA if necessary. 

Check compatibility of RNA with this assay. Pay particular attention to the presence of 

small RNAs before and after DNase treatment (using positive control such as 5S). 

RNase contamination 

Maintain aseptic conditions and use RNase decontamination solution, add RNase 

inhibitor. 

Some miRNA precursors may not be expressed in your sample. MiRNA precursor 

expression profiles vary from one tissue to another or the expression of miRNA 

precursors may be too low to be detected. 

Increase the amount of starting material (to do so in step 16 of the protocol dilute the 

cDNA in a lower final volume). 

> We recommend performing positive control reactions using U6, 5S, TBP and HMBS 

in parallel with your sample. 

SYBR ® Green I has degraded 

SYBR ® Green I should be protected from light whenever possible. 



16 

No product from experimental samples is amplified 

and positive controls work as expected 


expression profiles vary from one tissue to another or the expression of miRNA 

precursors may be too low to be detected 








Negative control is positive 

DNA or cDNA contamination 

Isolate source of contamination and replace reagent(s). Use separate pipettor for 

reaction assembly and post-PCR analysis. 

PCR products from previous PCR 

Add a UNG treatment, before starting your PCR program, to degrade contamination 

from previous PCR. 

Primer-dimer formation 

Check the Tm (normally product with lower Tm) to verify the primer-dimer formation. 

They are usually only detectable late in the cycling protocol (Ct > 40). 

No reverse transcriptase control is positive with a low CT 

The activity of the DNase is too low 

Check the date of expiration of your DNase or try with another batch. 





The dissociation curves does not show a specific peak 

RNA has been damaged or degraded 

Replace RNA if necessary. 






RNase contamination 

Maintain aseptic conditions and use RNase decontamination solution, add RNase 

inhibitor. 


expression profiles vary from one tissue to another or the expression of precursor 

miRNAs may be too low to be detected. 



> We recommend performing the positive control reactions with U6, 5S, TBP and 

HMBS in parallel with your sample. 

The dissociation curves shows unspecific peaks or several peaks 

Expression of a not-known miRNA 

Put the PCR product on agarose gel; purify and sequence the non-specific product. 




MgCl 2 concentration 

Optimize MgCl 2 concentration. 



18 

The Tm in the dissociation curve for a specific miRNA is different 

from one sample to another 

Variations in extraction methods 

Differences in Tm can be explained by differences in pH, salt or detergents in a 

reaction due to variations in different extractions. 

Modification of the same miRNA from one cell type to another 

It could be possible that a product of different size or small modifications of the same 

miRNA are obtained from one cell type to another. To determine which miRNA is 

amplified, PCR product should be cloned and sequenced. 

MiRNA Editing 

Different mechanism of maturation of pri-miRNA to pre-miRNA (miRNA editing) can 

occur from one cell type to another. To determine which miRNA is amplified, PCR 

product should be cloned and sequenced. 

Specificity of MirMaid miRNA set to precursor miRNA versus 

primary miRNA 

Sequences of pre-miRNAs are identical to sequences of pri-miRNAs. Then, the 

MiRmaid qRT-PCR primer set will amplify both the pri-miRNA and the pre-miRNA. 

Primary miRNAs are 10 to 100 times less abundant than pre-miRNAs. Thus, the 

amplification would be mainly reflecting the quantification of the precusor miRNAs. 

To obtain a precise quantification of the pre-miRNAs versus the pri-miRNAs, specific 

primers to pri-miRNAs can be designed and quantification of primary miRNAs can be 

subtracted; thus, precise expression rate of miRNA precursors will be obtained. 


Homology between human 

pre-miRNA primers and rodent 

sequences 

The table below presents the human pre-miRNA primer sets that could be used for the 

amplification of the mouse or rat miRNA precursors. When an amplification is obtained, 

the size of the PCR product is mentioned in the appropriated column. 

Table 3: Homology between human, mouse and rat pre-miRNAs 

PCR product size (bp) 

Mouse Rat 

mir 18 65 65 

mir 22 84 84 

mir 25 81 81 

mir 96 - 78 

mir 106b 66 66 

mir 103-2 54 54 

mir 152 - 64 

mir 103-1 55 55 

mir 132 59 59 

mir 150 - 58 

mir 221 75 75 

mir 223 65 65 

mir 369 60 60 

mir 377 64 - 

mir 379 61 - 

mir 382 71 71 

mir 224 - 82 

mir 323 75 75 

mir 30c1 52 52 

mir 10b - 71 


PCR product size (bp) 

Mouse Rat 

mir 125b1 - 66 

mir 128a 62 62 

mir 129-1 51 51 

mir 125b2 - 64 

let7a1 73 73 

mir 135b 77 77 

mir 138 2 69 69 

mir 153-2 - 62 

let7f1 68 68 

let7b 61 61 

let7 f2 70 70 

mir 19b1 58 58 

mir 200b - 80 

mir 23b 60 60 

mir 27a,b 66 66 

mir 29b2 61 61 

let7i 56 56 

mir 24-1 62 62 

mir 138-1,2 69 69 


20 

Guidelines for successful RT-qPCR 

The RT reaction should be set up in a clean environment to avoid contamination; cleaning 

solutions are available to avoid any RNase contamination. We also recommend working 

with RNase free plastics. The tubes containing the reaction should be maintained on ice 

during the set up of the reaction. 

When setting up a qPCR reaction, it is always recommended preparing a Reaction mix 

containing all the reagents required for the reaction in order to minimize the differences 

across the 96-well plate and allow for more accurate pipetting (as the volume required 

per sample is usually very small). The reaction mix should ideally be prepared in a 

separate room from the one where DNA samples have been prepared in order to avoid 

any contamination. 

The DNA samples should always be added to the side of each well before the reaction mix 

and then rinsed down while adding the reaction mix. As the reaction mix is heavier than 

the DNA, the DNA will be mixed within the reaction mix. The plate can gently be shaken 

on a plate shaker and spun before placing it on the machine. This is not an essential step 

but will ensure that the reactions are thoroughly mixed and collected at the bottom of the 

wells. It is useful to also check the wells for bubbles, as bubbles at the bottom of the well 

can produce unusual plots on the results, when using machines that read from the top. 

When sealing the PCR plates, it is important to ensure that it has been correctly done. 

If optical films are used, fingerprints and marks should be avoided on the top of the film. 

The qPCR machine used should be programmed according to the manufacturers 

instruction manual. 

The thermal cycling conditions described in this manual should be used as a good 

starting point, but optimization of these conditions may be required for certain assays to 

achieve optimal results. 


Ordering information 

Quantitative measurement of miRNA precursor expression level by 

Real-Time qPCR 

MiRmaid miRNA precursor qRT-PCR primer set (human) 

2 plates + 3 tubes RT-SYMI-025 

• MiRmaid miRNA precursor RT-qPCR primer set for 50 RT reactions (25 samples) 

• Forward and Reverse miRNA qPCR primers for 25 qPCR reactions 

+ qPCR MasterMix Plus for SYBR ® Green I for 600-25 μl reactions 

MiRmaid miRNA precursors qRT-PCR primer set (human) 

2 plates + 3 tubes RT-SYMI-025NR 

• MiRmaid miRNA precursor RT-qPCR primer set for 50 RT reactions (25 samples) 


+ qPCR MasterMix Plus for SYBR ® Green I No ROX for 600 - 25 μl reactions 

MiRmaid miRNA precursors qRT-PCR primer set (human) 

2 plates + 1 tubes RT-OLMI-025SET 

• MiRmaid miRNA precursor RT-qPCR primer set only for 50 RT reactions (25 samples) 


MiRmaid RT MasterMIX 1 tubes RT-OLRT-025MM 

• MiRmaid miRNA precursor RT primer MasterMIX for 50 RT reactions (25 samples) 

MiRmaid miRNA precursor qRT-PCR individual set 2 tubes RT-OLMI-XXXX* 

• MiRmaid miRNA RT primer (green cap) for 400 RT reactions 

• Forward and reverse miRNA qPCR primer mix (yellow cap) for 200 reactions (25 μl reaction) 

(*) Please find all unique identifier corresponding to each specific MiRmaid miRNA precursor qRT-PCR 

individual set in our website. 

MiRmaid miRNA precursor qRT-PCR control – U6 2 tubes RT-OLMI-U6 

• MiRmaid U6 RT primer for 400 RT reactions 

• Forward and reverse U6 qPCR primer mix for 200 reactions (25 μl reaction) 



22 

MiRmaid miRNA precursor qRT-PCR control – 5S 2 tubes RT-OLMI-5S 

• MiRmaid 5S RT primer for 400 RT reactions 

• Forward and reverse 5S qPCR primer mix for 200 reactions (25 μl reaction) 

MiRmaid miRNA precursor qRT-PCR control – HMBS 2 tubes RT-OLMI-HMBS 

• MiRmaid HMBS RT primer for 400 RT reactions 

• Forward and reverse HMBS qPCR primer mix for 200 reactions (25 μl reaction) 

MiRmaid miRNA precursor qRT-PCR control – TBP 2 tubes RT-OLMI-TBP 

• MiRmaid TBP RT primer for 400 RT reactions 

• Forward and reverse TBP qPCR primer mix for 200 reactions (25 μl reaction) 

Detection of mature miRNA expression level with MicroArray 

Human MiRanalyser LNA ® Array 5 arrays AR-MIRAH-01 

• Pre-printed Mature miRNA 

MiRanalyser MicroArray services service AR-PROF-01 

• The array, labelling of two RNA samples, hybridization and data analysis 

MiRNA Printing services service AR-GRD-GS 

• Printing of your predicted miRNA 

Human miRNA hybridization service service AR-HYMIC-01 

Human miRNA oligo set set AR-OLHM-SET 

Up-regulate gene expression using miRNA knockdown 

Clear-MiR miRNA inhibitors per target miRNA OR-0032 

• Synthesis scale: 0.2 μmol 

• Min. delivered quantity: 4 OD (+/- 20 nmol) 

Clear-MiR Plus miRNA inhibitors per target miRNA OR-0033 

• Synthesis scale: 0.2 μmol 

• Min. delivered quantity: 4 OD (+/- 20 nmol) 


Related products 

qPCR MasterMix plus for SYBR ® Green I 

The MiRmaid miRNA precursors qRT-PCR Primer Set (human) has been designed and 

validated on ABI Real Time thermocyclers and on MJ research (Biorad) Real-Time 

thermocyclers. However the MiRmaid miRNA precursor qRT-PCR Primer Set (human) 

could also be used on other platforms. 

Eurogentec can provide you with the correct kit according to the Real-Time thermocycler 

that will be used for this assay. Please find in find in appendix 3 the compatibility table 

relating each of our qPCR MasterMix for SYBR ® Green I for the use on each Real-Time 

thermocyclers. 

Uracil-N-Glycosylase 

Uracil-N-Glycosylase (300 U) 500 RXN 50 μl RT-0610-03 

Uracil-N-Glycosylase (1500 U) 2500 RXN 50 μl RT-0610-15 

DNase 

DNase 1 mg ME-0170-10 

dNTP Set 

dNTP Set (4 x 5 μmoles) 4 x 500 μl NU-0020-10 



24 

Appendix 1: Layout of MiRmaid qPCR primer plate #1 

A 

B 

C 

D 

E 

F 

G 

H 

1 2 3 4 5 6 7 8 9 10 11 12 

mir 

18a 

mir 

107 

mir 

151 

mir 

220 

mir 

224 

mir 

325 

mir 

384 

mir 

506 

mir 

20a 

mir 

155 

mir 

221 

mir 

296 

mir 

330 

mir 

425 

mir 

507 

mir 

21 

mir 

103-1 

mir 

182 

mir 

223 

mir 

301 

mir 

338 

mir 

429 

mir 

508 

mir 

22 

mir 

132 

mir 

184 

mir 

369 

mir 

323 

mir 

490 

mir 

431 

mir 

522 

mir 

25 

mir 

30b 

mir 

190 

mir 

370 

mir 

30C1 

mir 

26b 

mir 

432 

mir 

525 

mir 

28 

mir 

140 

mir 

193 

mir 

371 

mir 

26a-2 

mir 

520h 

mir 

452 

pri 

mir 

9-3 

Appendix 1: Layout of MiRmaid qPCR primer plate #2 

A 

B 

C 

D 

E 

F 

G 

H 

Manual for MiRmaid miRNA precursor qRT-PCR Primer Set I www.eurogentec.com I info@eurogentec.com 

mir 

93 

mir 

141 

mir 

205 

mir 

372 

mir 

1-1 

mir 

491 

mir 

453 

mir 

527 

mir 

95 

mir 

144 

mir 

206 

mir 

374 

mir 

340 

mir 

493 

mir 

455 

pri 

mir 

106a 

mir 

96 

mir 

146a 

mir 

208 

mir 

377 

mir 

520c 

mir 

494 

mir 

484 

U6 

mir 

106b 

mir 

147 

mir 

181a-1 

mir 

379 

mir 

361 

mir 

495 

mir 

485 

5S 

mir 

103-2 

mir 

150 

mir 

215 

mir 

380 

mir 

367 

mir 

498 

mir 

487a 

1 2 3 4 5 6 7 8 9 10 11 12 

mir 

10b 

mir 

135a1 

mir 

181b2 

mir 

19b1 

mir 

365-1 

mir 

518c 

mir 

7-3 

mir 

122a 

let 

7a1 

pri 

mir 

186 

mir 

181b1 

mir 

376b 

mir 

519a 

1,2,e 

pri 

let 

7a-3 

mir 

125b1 

mir 

135b 

let 

7b 

mir 

200b 

mir 

26a-1 

mir 

521-1 

mir 

194-1 

mir 

128a 

mir 

138-2 

mir 

365-2 

mir 

218-1,2 

mir 

30d 

mir 

526b 

mir 

346 

mir 

129-1 

pri 

mir 

142 

mir 

196a2 

mir 

23b 

let 

7d 

mir 

518f 

mir 

371 

mir 

125b2 

mir 

148b 

pri 

let 

7c 

mir 

517a 

mir 

422a 

mir 

24-1 

mir 

98 

mir 

133a 

1,2 

mir 

135a2 

mir 

199a1 

mir 

101-2 

let 

7i 

mir 

521-2 

mir 

138-1,2 

mir 

15a 

mir 

153-2 

pri 

mir 

19a 

mir 

515-1,2 

pri 

mir 

31 

mir 

let 

7a2 

mir 

219-1 

mir 

129-2 

let 

7f1 

mir 

302d 

mir 

516-3,4 

pri 

mir 

326 

mir 

218-1 

U6 

mir 

130a 

mir 

15b 

let 

7 f2 

mir 

27a 

pri 

mir 

331 

mir 

7-1 

5S 

TBP 

mir 

181a 

mir 

302c 

mir 

29b2 

mir 

34a 

mir 

92-1 

TBP 

mir 

152 

mir 

154 

mir 

217 

mir 

382 

mir 

520g 

mir 

501 

mir 

488 

HMBS 

mir 

133b 

mir 

19b2 

mir 

450-1 

mir 

518a2 

mir 

99a 

mir 

196b 

HMBS 

RT 

Master 

MIX

Appendix 3: qPCR MasterMixes compatibility with Real-Time thermocyclers 

ABI GeneAmp ® SDS 5700 iCycler iQ ® Mx4000 ® Mini Opticon ® 

ABI Prism ® SDS 7000/ iQ5 Mx3000P ® DNA Engine Opticon ® 1 

7300/7700/7900HT ABI Prism ® 7500* My iQ Mx3005P ® DNA Engine Opticon ® 2 

MasterCycler ® ep RealPlex* Chromo 4 

Quantica ® 

LC480* 

With ROX passive reference Reference # RXN wwiitthh RROOXX ww//oo RROOXX 

qPCR MasterMix Plus for SYBR ® Green I w/o UNG RT-SN2X-03WOU+ 300 ✔ 

qPCR MasterMix Plus for SYBR ® Green I dTTP RT-SN2X-03+WOUN 300 ✔ 

qPCR MasterMix for SYBR ® Green I • 5 x 1.5 ml RT-SN2X-03T 300 ✔ 

qPCR MasterMix Plus for SYBR ® Green I • 7.5 ml RT-SN2X-03+ 300 ✔ 

qPCR MasterMix Plus for SYBR ® Green I • 15 ml RT-SN2X-06+ 600 ✔ 

qPCR MasterMix Plus for SYBR ® Green I • 50 ml RT-SN2X-20+ 2000 ✔ 

qPCR MasterMix Plus for SYBR ® Green I Low ROX RT-SN2X-03+WOULR 300 ✔ ✔ 

Without ROX passive reference 

qPCR MasterMix Plus for SYBR ® Green I No ROX RT-SN2X-03+NR 300 ✔ ✔ 


qPCR MasterMix Plus for SYBR ® Green I with fluorescein RT-SN2X-03+WOUFL 300 ✔ 


26 

Trademarks 

ABI Prism ® is a registered trademark of The Perkin-Elmer Corp. 

DNA Engine Opticon ® is a registered trademark of MJ Research, Inc. 

Eppendorf ® is a registered trademark of Eppendorf AG. 

Eurogentec ® is a registered trademark of Eurogentec S.A. 

GeneAmp ® is a registered trademark of Roche Molecular Systems, Inc. 

iCycler iQ ® is a registered trademark of Bio-Rad Laboratories, Inc. 

iCycler ® is a registered trademark of Bio-Rad Laboratories, Inc. 

MasterCycler ® is a registered trademark of Eppendorf, Inc 

Mx3000 ® is a registered trademark of Stratagene 

Mx3000P ® is a registered trademark of Stratagene 

Mx4000 ® is a registered trademark of Stratagene 

Quantica ® is a registered trademark of Techne Ltd and Techne, Inc. 

Smartcycler ® is a registered trademark of Cepheid, Inc. 

SYBR ® Green is a registered trademark of Molecular Probes, Inc. 

Disclaimer 

Chemically-Modified Hot-Start Polymerase Reagents and Kits 

Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155, 5,677,152, 5,773,258, 

and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims 

for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim (such as the patented 5’ Nuclease Process claims in US Patents Nos. 

5,210,015 and 5,487,972) and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial 

consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further 

information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. 

Licensed dsDNA-Binding Dye Research Kits 

Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155, 5,677,152, 5,773,258, 

5,407,800, 5,322,770, 5,310,652, 5,994,056, 6,171,785, and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable 

immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim (such as apparatus 

or system claims in US Patent No. 6,814,934) and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a 

fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate 

license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 

94404, USA. 

UDG 

Use of UDG employs U.S. Patents 5,035,996, 5,945,313, 5,683,896 and their foreign counterparts licensed to Eurogentec S.A. from Invitrogen Corporation. 

Cyclic-substituted unsymmetrical cyanine dyes 

Cyclic-substituted unsymmetrical cyanine dyes are covered by U.S. Patents 5,436,134 and 5,658,751 and licensed to Eurogentec S.A. by Molecular Probes, Inc. in the direct research field. 


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Manual for MiRmaid miRNA precursor qRT-PCR Primer Set I www.eurogentec.com info@eurogentec.com 27 

I

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