RNA was extracted from 800 µl plasma using an miRNeasy serum/plasma kit (Qiagen China, Co., Ltd., Shanghai, China) according to the manufacturer's instructions. The RNA was eluted in 50 µl nuclease free water and 40 ng RNA (in 5 µl) was reverse transcribed with TaqMan® MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. cDNA was quantitated using a TaqMan-based RT-qPCR assay specific for miR-208a and miR-370 according to the manufacturer's recommended protocol on an Applied Biosystems 7500 Sequence Detection System (the miR-208a and miR-370 specific primers for RT and PCR were enclosed in the kits). The cycling conditions were 95°C for 10 min followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. All reactions were performed in triplicate. The expression levels of circulating miRNAs are presented as the quantitation cycle (Cq) values. Exogenously added cel-miR-39 served as a control for normalizing the data. The comparative Cq (ΔCq) method was used to analyze the miRNA expression levels (15 (link)).
Protocol detail
Quantification of Circulating miRNAs
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Corresponding Organization :
Other organizations : Affiliated Hospital of Jining Medical University, Dalian Medical University
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Variable analysis
independent variables
- None explicitly mentioned
- Expression levels of circulating miRNAs (miR-208a and miR-370)
- RNA extraction method (miRNeasy serum/plasma kit)
- Reverse transcription method (TaqMan® MicroRNA Reverse Transcription kit)
- RT-qPCR assay (TaqMan-based)
- Thermal cycling conditions (95°C for 10 min, then 40 cycles of 95°C for 15 sec and 60°C for 1 min)
- Normalization using exogenously added cel-miR-39
- Positive control: None mentioned
- Negative control: None mentioned
- Exogenously added cel-miR-39 served as a control for normalizing the data
Annotations
Based on most similar protocols
The Input protocol and protocols 1-5 all use TaqMan-based RT-qPCR assays for quantification of circulating miRNAs. This approach provides high specificity and sensitivity for accurate miRNA detection and quantification. (Input protocol, Protocol 1, Protocol 3, Protocol 4, Protocol 5)
Exogenously added cel-miR-39 is used as a spike-in control across multiple protocols to normalize the data and account for variations in RNA extraction and RT-qPCR efficiency. This ensures accurate quantification of the target miRNAs. (Input protocol, Protocol 1, Protocol 3, Protocol 4, Protocol 5)
The Input protocol and Protocol 3 both use the miRNeasy Serum/Plasma kit for RNA extraction, which is a well-established and reliable method for purifying miRNAs from plasma or serum samples. This helps ensure consistent and high-quality RNA isolation. (Input protocol, Protocol 3)
The Input protocol and Protocol 2 both measure the RNA quality by evaluating the 260/280 nm absorbance ratio, ensuring that only high-quality RNA samples (ratio > 1.8) are used for downstream analysis. This step helps eliminate potential PCR inhibitors and ensures reliable miRNA quantification. (Input protocol, Protocol 2)
The Input protocol and Protocol 3 both use TaqMan Advanced miRNA Assays, which provide enhanced sensitivity and specificity for miRNA detection compared to traditional TaqMan assays. This allows for accurate quantification of low-abundance miRNAs. (Input protocol, Protocol 3)
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