An evaluation of the applicability of microarrays for monitoring toxic algae in Irish coastal waters

An assessment of RNA content inPrymnesium parvum,Prymnesium polylepis,cf.Chattonellasp. andKarlodinium veneficumunder varying environmental conditions for calibrating an RNA microarray for species detection

“…The clade‐level probes Clade01old_25_dT for Prymnesium target species contain the poly‐T tail but was not extended by 25 nucleotides and is the same original sequence from Simon et al . () and was renamed Clade01 in the second‐generation chip (McCoy et al ., ). However, given that the probe still produces a good signal for Prymnesium targets, it was included on the third‐generation chip.…”

“…The P. parvum species-specific probe PparvD01_25_dT originally called PRYM694 was not extended by 25 nucleotides as the name suggests and is still the original sequence plus the addition of a 15-nucleotide poly-T tail from the second-generation chip (McCoy et al, 2013). This may explain the poor performance of the PparvD01_25_dT probe compared to the other P. parvum-specific probe Prymparv01_25_dT, which was redesigned from the genus-level probe PrymGS01_25 from the second-generation chip (Eller et al, 2007).…”

“…This may explain the poor performance of the PparvD01_25_dT probe compared to the other P. parvum-specific probe Prymparv01_25_dT, which was redesigned from the genus-level probe PrymGS01_25 from the second-generation chip (Eller et al, 2007). It was established that the PrymGS01_25 probe had more affinity towards P. parvum species when extended by 25 nucleotides plus poly-T tail on the third-generation chip (McCoy et al, 2013). The clade-level probes Cla-de01old_25_dT for Prymnesium target species contain the poly-T tail but was not extended by 25 nucleotides and is the same original sequence from Simon et al (1997) and was renamed Clade01 in the second-generation chip (McCoy et al, 2013).…”

“…It was established that the PrymGS01_25 probe had more affinity towards P. parvum species when extended by 25 nucleotides plus poly-T tail on the third-generation chip (McCoy et al, 2013). The clade-level probes Cla-de01old_25_dT for Prymnesium target species contain the poly-T tail but was not extended by 25 nucleotides and is the same original sequence from Simon et al (1997) and was renamed Clade01 in the second-generation chip (McCoy et al, 2013). However, given that the probe still produces a good signal for Prymnesium targets, it was included on the third-generation chip.…”

“…detection limits were determined from the second-generation microarrays at levels > 5 ng of RNA, which approximately corresponds to 8800 cells of P. parvum and 3800 cells of P. polylepis. The only difference between the second-and third-generation microarray was that probes that showed nonspecificity in the former were omitted from the latter (McCoy et al, 2013). The slopes of the calibration curves generated using varying amounts of labelled RNA were then used to infer cell numbers from the microarray signal intensities via a GPR-ANALYZER program (Dittami & Edvardsen, 2013).…”