Products

miRNA qRT-PCR Primers

Description：

Ribobio"s Bulge-Loop™ miRNA qRT-PCR Primer Set consists of a specific RT primer, forward primer, and a universal reverse primer to any mature miRNA sequences. All the specificity designed maximally meets various needs of your owned miRNA qRT-PCR assay.

- specific design

- Accurate quantification for less than 10 copies of miRNAs

- Reliable performace for standard protocol

- Fast and easy to use

- Thousands options of miRNA sets for human, mouse and rat

No Data!

Click, to search more products >>

Bulge-Loop™ miRNA qRT-PCR is a RiboBio’s proprietary assay developed for detection and quantitation of mature miRNAs using SYBR Green I. It is highly sensitive and specific to sequence of interest, while no fluorescently labeled probe is needed. Bulge-Loop™ miRNA qRT-PCR Primer Set consist of for quantitative PCR. Enzymes that are required for the RT and PCR reaction are not provided.

Table 1. Constitutes of Bulge-Loop™ miRNA qRT-PCR Primer Sets

Reagent	100 rxns	200 rxns	500 rxns	1000 rxns	Storage
Bulge-Loop™ RT Primer	1 nmol	1 nmol	1 nmol	1 nmol	-20℃
Bulge-Loop™ Forward Primer	1 nmol	2 nmol	5 nmol	10 nmol
Bulge-Loop™ Reverse Primer	1 nmol	2 nmol	5 nmol	10 nmol

Bulge-Loop™ miRNA qRT-PCR is a RiboBio’s proprietary assay developed for detection and quantitation of mature miRNAs. It includes two steps: 1) the reverse transcription using a specially designed bulge-looped primer and 2) the real-time PCR amplification using SYBR Green reagent. No fluorescently labeled probe is needed. The reverse transcription primer with the secondary structure is ligated to the 3’ end of miRNA, initiating the cDNA synthesis by reverse transcrptase. Quantitative measurement is carried out by real time PCR using miRNA-specific forward primer and general reverse primers.

Figure 1. Bulge-Loop™ miRNA qRT-PCR.

Bulge-Loop™ miRNA qRT-PCR primer set for human, mouse and rat miRNAs are available in stock. Custom primers for other species will be provided upon request.

Quantification by Bulge-Loop miRNA qRT-PCR can be accurate within a broad 107-fold dynamic range. You can detect miRNAs either as low as 5 copies in 0.1pg of total RNAs, or highly abundant miRNAs in 1µg total RNAs with high sensitivity and accuracy.

Specificity

The RiboBio’s Bulge-Loop™ qRT-PCR based approach demonstrates enhanced specificity and allows detection and differentiation of highly homologous miRNAs, as demonstrated below using as example the hsa-let-7 family.

The hsa-let-7 miRNA family is composed of highly conserved miRNAs, which differ from each other only by a few nucleotides. It is impossible to differentiate such homologous molecules using traditional detection methods.

Table 2. Sequence comparison of hsa-let-7 family: the varied nucleotides are indicated in red.

Sensitivity

Bulge-Loop miRNA qRT-PCR has a broader 107-fold dynamic range than other detection assay such as northern blotting and microarray; is able to detect miRNAs as low as 5 copies in 0.1pg of total RNAs.

Figure 2. Amplification curves of series dilution of a synthetic miRNA molecule.

Accuracy

The expression levels of miRNAs can be precisely measured from minimum total RNA input by using Bulge-Loop miRNA qRT-PCR, so it can be used for validating the miRNA microarray data.

Figure 3. The dynamic range of Bulge-loop miRNA qRT-PCR

Seven synthetic miRNAs corresponding to each of the has-let-7 family members were mixed together at equimolar amounts and subjected to quantitative RT-PCR using Bulge-Loop reagents. The results demonstrate that miRNA-specific primers allow to clearly differentiate seven individual members of the hsa-let-7 miRNA family.

Table 3. Sentivity of Bulge-Loop™ miRNA qRT-PCR Primer Sets for hsa-let-7 miRNA family.

Shipping

Shipped in room temperature as powder.

Storage

Store at -20°C to -80°C. Stable for one year.

Resuspension

1. Briefly centrifuge tubes to ensure that the pellet is collected at the bottom of the tube.

2. Resuspend primer to a convenient stock concentration using 200μl RNase-free water for stock solution of 5mM concentrition.

4. Briefly centrifuge tubes to ensure that the solution is collected at the bottom of the tube.

5. Verify the concentration of primer using UV spectrophotometry (at 260 nm).

6. Divide into several tubes, and store at -20ºC to -80ºC.

For best results, limit freeze-thaw events of each tube to no more than five.

miRNA qRT-PCR

The following procedures are given as a reference protocol. Please refer to other technical resources for further information.

1) miRNA RT Reaction

1. Dilute 1 μl of 5mM RT primer in 79 μl RNase-free H2O, to make the 62.5 nM RT primer working solution.

2. 50μl RT reaction is recommended.

Reagent	25 μl reaction	50μl reaction	Final concentration
RNA template (2µg)	x μl	x μl
RT primer (62.5 nM)	2 μl	4 μl	5 nM
RNase-free H₂O	Up to 11 μl	Up to 19 μl

Notes: The optimal final concentration of RT primers ranges from 5~50nM.

Mix the components above well, briefly centrifuge and incubate at 70℃ for 10 minutes. Place on ice for 2mins, and add the following components. Reverse transcription may be carried out in a thermal cycler, incubating at 42℃ for 60 min, 70℃ for 10 min.

Reagent	25 μl reaction	50μl reaction	Final concentration
RT buffer 5X	5 μl	10 μl	1X
dNTP mix (2.5mM)	2 μl	4 μl	0.2 mM
RNase inhibitor (40U/mL)	0.5 μl	1 μl
Reverse transcriptase (200U/mL)	0.5 μl	1 μl
RNase-free H₂O	Up to 25 μl	Up to 50 μl

Notes: cDNA should be immediately placed on ice after reverse transcription. The following procedures may be carried out on ice.

2) miRNA qPCR reaction

1. Assemble the SYBR Green Mix: including Taq DNA polymerase, dNTP mix, PCR Buffer, SYBR GreenⅠ.

Recommended reaction setup for Bulge-Loop™ qRT-PCR:

Reagent	25 μl reaction	50μl reaction	Final concentration
SYBR Green Mix	9 μl	22.5 μl	1X
RT product	2 μl	2 μl
Bulge-Loop™ miRNA Forward Primer (5 mM)	2 μl	5 μl	500 nM
Bulge-Loop™ miRNA Reverse Primer (5 mM)	2 μl	5 μl	500 nM
RNase-free H₂O	Up to 25 μl	Up to 50 μl

2. Program the fast-capable real-time PCR instrument (e.g. Bio-Rad CFX96) as shown in the following table. 3-step PCR is recommended：

Cycles	Steps	Temperature	Time	Purpose
1Ｘ	Initiation	95℃	20 sec	Denaturing the cDNA template
40Ｘ	Denaturation	95℃	10 sec	Denaturing the PCR products
	Annealing	60℃	20 sec	Primers annealing to the template
	Elongation	70℃	1-10 sec	PCR product amplification

3. For qPCR reaction utilizing SYBR GreenⅠas the dsDNA intercalating reagent, melting curve analysis may be desirable following the PCR cycling, with the melting performed from 70 to 95 ℃ at0.4℃/s melt rates (holding for 1 second).

Sign In | Register

miRNA qRT-PCR Primers

Description：

Click, to search more products >>