Effect of buffer composition on PNA–RNA hybridization studied in the microfluidic microarray chip

Abstract

Herein, we report that peptide nucleic acid sequences (PNAs) have been used as the probe species for detection of RNA and that a microfluidic microarray (MMA) chip is used as the platform for detection of hybridizations between immobilized PNA probes and RNA targets. The RNA targets used are derived from influenza A sequences. This paper discusses the optimization of two probe technologies used for RNA detection and investigates how the composition of the probe buffer and the content of the hybridization solution can influence the overall results. Our data show that the PNA probe is a better choice than the DNA probe when there is low salt in the probe buffer composition. Furthermore, we show that the absence of salt (NaCl) in the hybridization buffer does not hinder the detection of RNA sequences. The results provide evidence that PNA probes are superior to DNA probes in term of sensitivity and adaptability, as PNA immobilization and PNA–RNA hybridization are less affected by salt content in the reaction buffers unlike DNA probes.

Necessity and Challenges of Sample Preconcentration in Analysis of Multiple MicroRNAs by Capillary Electrophoresis

“…It is known that low-concentration buffers are typically not favored by nucleic acid hybridization . However, thanks to the neutral backbone of PNA, PNA–RNA duplexes are much more stable than the DNA–RNA duplexes in low-concentration buffers due to the lack of Coulomb repulsion forces between the two strands . Relative neutrality of PNA with respect to DNA should make it possible to perform PNA–miRNA hybridization reactions in low-concentration buffers.…”

“…33 However, thanks to the neutral backbone of PNA, PNA−RNA duplexes are much more stable than the DNA−RNA duplexes in low-concentration buffers due to the lack of Coulomb repulsion forces between the two strands. 34 Relative neutrality of PNA with respect to DNA should make it possible to perform PNA−miRNA hybridization reactions in low-concentration buffers. To confirm this, we prepared hybridization mixtures in Tris−Cl buffers with concentrations of 100, 10, and 1 mM.…”

Nucleic acid amplification test (NAAT) conducted in a microfluidic chip to differentiate between various ginseng species

“…The genotype of the SNP site is then identified by introducing ddNTPs, each labelled with a different fluorophore. 16 Many microfluidic DNA detection methods have been developed; 39 these include methods for probe-PCR strand hybridization, [17][18][19][20][21][22][23] qPCR, [24][25][26] sequencing, 27,28 etc. A major issue these techniques have is that they all require PCR amplification which in turn requires a thermocycler.…”

“…Many microfluidic DNA detection methods have been developed; 39 these include methods for probe-PCR strand hybridization, 17–23 qPCR, 24–26 sequencing, 27,28 etc . A major issue these techniques have is that they all require PCR amplification which in turn requires a thermocycler.…”