TABLE 7

Effects of Anticoagulants or Processing Techniques on Nucleic Acid Isolation

Sample TypeEffects on RNA and DNA
RNA
 Effect on stability and quality• PAXgene tubes: produce high-quality RNA due to both rapid stabilization of RNA during sample collection and to removal of hemoglobin and plasma proteins90
 Effect on functional studies• EDTA: produces highly fragmented and degraded RNA,140 increases the levels of 5′ RNA tags quantified by qPCR due to dead leukocytes,141 but has no effect on RNA integrity and quantity when samples undergo 1 freeze-thaw process;140 although RNA is degraded in EDTA, ≥310-bp products can be amplified140
 Effect on sample handling or collection• BD Vacutainer CPT cell preparation tube with sodium citrate or heparin: produces a significantly higher number of copies of RNA of HIV virus as compared with standard Vacutainer tubes at 24 and 72 h after the blood samples are drawn142
• PAXgene tubes: suitable for gene expression analyses, hybridizations, microarrays, and kinetic reverse transcription-PCR assays90
• BD Vacutainer CPT cell preparation tube with citrate or jeparin: useful for measuring virion RNA levels with reverse transcription-PCR-ELISA and branched DNA assay142
• EDTA: useful to isolate circulating RNA in plasma but requires large volumes of sample and a plasma concentration by evaporation step at 4°C140
• PAXgene tubes: samples cannot be stored at RTa for >72 h, or at 4°C for >6 d before processing90
DNA
 Effect on stability and quality• Citrate and EDTA: optimal choices to isolate cell-free DNA57
 Effect on functional studies• PAXgene tubes: prevent blood coagulation and stabilize leukocytes143
 Effect on sample handling or collection decisions• EDTA: can alter Mg2+ concentrations and interfere with cytogenetic analyses25,26
• Heparin: should be avoided, because it can inhibit downstream PCR reactions89
• DNA extraction method using dried blood spots on filter paper provides a reliable molecular diagnosis of viral and parasite infections50,144
• DNA from whole-blood samples should not be frozen before DNA isolation, because hemolysis can inhibit PCR reactions58
• PAXgene tubes: allow for isolation of highly pure genomic DNA suitable for PCR, quantitative real-time PCR, Southern blotting, SNP genotyping, and pharmacogenomic studies143
• PAXgene tubes: fragments isolated range from 20 to 200 kb in size, with an average length of 50–150 kb; blood samples are stable when stored in the tubes for ≤14 d at RT143
  • BD, BD Biosciences; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; SNP, single nucleotide polymorphism.

  • a RT = 20–25°C.