This kit provides a fast, reliable and convenient method to purify and concentrate high quality, high purity and inhibitor-free cell-free circulating DNA, circulating including exosomal RNA as well as viral DNA/RNA from fresh or frozen serum or plasma samples prepared from blood collected on either EDTA or Citrate from volumes ranging from 2 mL to 5 mL. Plasma samples prepared from blood collected on heparin should not be used as heparin can significantly interfere with many downstream applications such as RT-PCR
The first column will handle the large volume input of bodily fluids that is followed by a concentration on a mini column for a final elution of 50 µL to 100 µL. All components for the purification are provided in one convenient and fast kit for the easy processing of small input volumes of bodily fluids. For a schematic workflow of the protocol click here.
The purified plasma/serum circulating and viral nucleic acid is fully compatible with all downstream applications including PCR, qPCR, methylation-sensitive reverse transcription qPCR, reverse transcription PCR, methylation-sensitive PCR and Southern Blot analysis, Northern blotting, RNase protection and primer extension, expression array assays, and NGS.
Plasma/serum cell-free circulating and viral nucleic acid has the potential to provide biomarkers for certain cancers and disease states as well as for the detection of viral infection. Currently, significant advancements are being made in utilizing cfc-DNA as biomarkers for the early diagnosis, prognosis and monitoring of therapy for several cancer types and autoimmune diseases. Cell-free mitochondrial DNA (cfmtDNA) is also under investigation for its clinical significance. This cfc-DNA is usually present as short fragments of less than 1000 bp. In addition, cell-free fetal DNA has been widely used as a non-invasive method for prenatal diagnosis including early identification of fetal sex, genetic studies for families at high risk for inherited genetic disorders. screening for Rhesus factor, screening for aneuploidy and identification of preeclampsia Exosomes are 40 - 150 nm membrane vesicles, which are secreted by most cell types. Exosomes can be found in saliva, blood, urine, amniotic fluid and malignant ascitic fluids, among other biological fluids. Evidence has been accumulating recently that these vesicles act as cellular messengers, conveying information to distant cells and tissues within the body. These exosomes may play a functional role in mediating adaptive immune responses to infectious agents and tumours, tissue repair, neural communication and transfer of pathogenic proteins. For this reason exosomal RNAs may serve as biomarkers for various diseases including cancer. As the RNA molecules encapsulated within exosomes are protected from degradation by RNAses they can be efficiently recovered from biological fluids, such as plasma or serum.