Total RNAs from tissues or cells were isolated using the miRNeasy Kit (Qiagen, Germantown, MD), followed by reverse transcription and real-time quantitative RT-PCR as described in our previous publication (27 (link)). Relative quantities of miRNAs were calculated using the 2−ΔΔCt method (28 (link)) with U6 snRNA (Applied Biosystems) as the endogenous control. RNAs from sera were isolated with miRNeasy Serum/Plasma Kit in conjunction with the synthetic spike-in control (cel–miR-39 mimic; Qiagen) for internal normalization. Quantitative detection of mRNA transcripts was carried out by real-time PCR with SYBR Green PCR mix (Applied Biosystems). Results were normalized to mRNA levels of β-actin. The primers used are given in Supplementary Tables 1 and 2 .
Protocol detail
Quantitative Analysis of miRNAs and mRNAs
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Actin
Cells
Mirnas
Mrna
Plasma
Primers
Real time pcr
Reverse transcription
Rnas
Serum
Sybr green
Tissues
U6 snrna
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Variable analysis
independent variables
- None explicitly mentioned
- Relative quantities of miRNAs
- Quantitative detection of mRNA transcripts
- U6 snRNA (Applied Biosystems) as the endogenous control
- Synthetic spike-in control (cel–miR-39 mimic; Qiagen) for internal normalization of miRNAs from sera
- β-actin mRNA levels for normalization of mRNA transcripts
- Positive control: U6 snRNA (Applied Biosystems) and β-actin mRNA levels
- Negative control: Synthetic spike-in control (cel–miR-39 mimic; Qiagen) for miRNAs from sera
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