0.5 µg total RNA was reversely transcribed into cDNA using the miScript II RT kit (Qiagen, Netherlands). Then, cDNA was mixed with miScript SyBR green qPCR mastermix (Qiagen). 25 µl of the cocktail was aliquoted into each well of 96 well plates containing the pre-dispensed miRNA-specific assays [Human Serum/plasma miRNA PCR Array, SABiosciences (Qiagen)]. PCR and data analysis was performed on an ABI Prism 7300 system (Applied Biosystems, USA). The transcript abundance was expressed as fold change over the value of the healthy control group calculated by 2−∆∆Ct method. Eight housekeeping genes including miR-39 and U6 were used. Since U6 was quite consistent among the three groups among eight housekeeping genes, U6 was chosen as the reference miRNA for validation study by real-time PCR.
Human serum plasma mirna pcr array
Manufactured by Qiagen
The Human Serum/plasma miRNA PCR Array is a laboratory equipment product designed for the quantitative analysis of microRNA (miRNA) expression in human serum or plasma samples. The core function of this array is to facilitate the simultaneous detection and measurement of multiple miRNA targets in a single experiment.
Automatically generated - may contain errors
Lab products found in correlation
Miscript 2 rt kit, by Qiagen (2 mentions)
Abi prism 7300 system, by Thermo Fisher Scientific (1 mentions)
Miscript sybr green qpcr mastermix, by Qiagen (1 mentions)
Steponeplus machine, by Thermo Fisher Scientific (1 mentions)
Mirneasy serum plasma extraction kit, by Qiagen (1 mentions)
Ingenuity pathway analysis software, by Qiagen (1 mentions)
Qiazol mirneasy kit, by Qiagen (1 mentions)
3 protocols using human serum plasma mirna pcr array
1
Quantitative Profiling of Serum/Plasma miRNAs
2
miRNA Profiling of Serum/Plasma Samples
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Total RNA was extracted with the miRNeasy Serum/Plasma extraction kit (Qiagen, Redwood City, CA) and purified using RNeasy MinElute Spin Columns. A total of 250 ng RNA was then reverse transcribed with a miScript II RT kit (Qiagen). miRNA expression profile was then measured using Qiagen’s Human Serum & Plasma miRNA PCR Array. Quantitative PCR was performed using a StepOnePlus machine (Applied Biosystems) per the manufacturer’s instructions. miRNA array data were analyzed using Ingenuity pathway analysis software (Qiagen) and DIANA-mirPath.
3
Extracting and Analyzing miRNA from Plasma
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Total RNA, including the miRNA fraction, was extracted from both plasma and supernatants obtained from in vitro studies by using the QIAzol miRNeasy kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions13 (link) (Online Supplementary Appendix). To identify the changes that occurred in the expression levels of miRNAs in plasma from APS patients and HDs, a Human Serum & Plasma miRNA PCR-array (Qiagen) was performed (Online Supplementary Appendix) on an exploratory cohort (Online Supplementary Table S2).
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