A simplified protocol for profiling heparin-contaminated circulating miRNAs: by microfluidic array.
Mol Biol Rep
; 47(12): 9973-9977, 2020 Dec.
Article
em En
| MEDLINE
| ID: mdl-33170428
ABSTRACT
Peripheral blood is a valuable, non-invasive source of biomarkers which include circulating miRNAs. Using microfluidic array-based techniques, miRNAs can be successfully measured in small amounts of blood plasma (< 0.5 mL) using cDNA pre-amplification. However, the use of heparin-based anticoagulants for blood collection hinders the detection of circulating miRNAs due to its inhibitory effect on PCR components. Although pre-treatment with heparinase have been shown to overcome heparin contamination in blood, its effect has not been described in array-based analyses or more sensitive applications with smaller sample volumes (i.e. 200 µL plasma) requiring pre-amplification. We show that the treatment of miRNA extracted from heparinised plasma with an optimised concentration of Bacteroides heparinase I prior to cDNA pre-amplification dramatically improves the number of detectable miRNA from 2 to 67 targets on the TaqMan® Array Human MicroRNA Cards. Furthermore, the titrated amount of heparinase (3 U) gave the best miRNA detection compared to those used in previous studies (6-24 U). This study provides novel data which demonstrates that heparinase treatment is compatible with protocols that involve pre-amplification of cDNA and microfluidic array-based techniques. This an improved methodology that permits miRNA-based biomarker analysis from small volume of heparinised plasma.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Manejo de Espécimes
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Heparina Liase
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MicroRNAs
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Microfluídica
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article