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RiboBio has developed a proprietary Bulge-loop™ miRNA qRT-PCR technology for detection of mature miRNAs. The quantitative assay uses the SYBR Green reagent, rather than conventional fluorescent probe, and is highly sensitive, and specific.

1. Bulge-Loop™ miRNA qPCR service includes:

a) Relative quantitation: Examine the expression of relative miRNA using U6 snRNA or 5S rRNA as internal controls

b) Absolute quantitation: chemically synthesized miRNA mature strand (MQP-0412) is used to plot the standard curve. At least 5-point series dilution of the synthetic miRNA will be measured, and the absolute abundance of miRNA is determined accordingly.

c) Confirmation of miRNA microarray data: qRT-PCR is an important tool to validate the expression level of selected miRNA microarray hits.

2 .Kit and equipment required for Bulge-Loop™ miRNA qPCR:

a) SYBR Green I quantitative PCR kit

b) Bio-Rad CFX96, ABI7300 etc.

3. Brief experimental procedures of Bulge-Loop™ miRNA qPCR:

a) RNA sample preparation

Total RNA samples should be provided by customers, extra fee may be applied if cultured cells, tissues or blood samples are submitted.

b) RNA quality test

A260 and A280 value are measured by spectrophotometer, concentration and purity are examined by agarose electrophoresis.

c) Reverse transcription

cDNA is synthesized by bulge-loop primers

d) Real-time PCR

Quantitative amplification with SYBR Green (triplet repeats for each sample), and U6 snRNA or 5S rRNA will be used as internal controls (loading control)

e) Data analysis and experimental reports

Relative quantification: Analysis of relative expression parameters, statistical analysis, so as to obtain the relative expression differences

Absolute quantification: The standard curve is made according to the miRNA standard production (at least five data points on each curve) so as to quantitatively detect the miRNA sample

We will analyze the real-time quantitative PCR results and provide you with the real-time quantitative PCR experimental data, amplification curve, dissociation curve, and bar graph.

Figure 1. The amplification curve detected by Bulge-Loop™ miRNA qRT-PCR.

Figure 2. The melting curve detected by Bulge-Loop™ miRNA qRT-PCR.

Figure 3. Expression differences of hsa-miR-122 in 5 samples analyzed by Bulge-Loop™ miRNA qRT-PCR.

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