Quantstudio 6 flex real time pcr detection system

Cells from overnight cultures of V. harveyi or A. fischeri were pelleted by centrifugation at 21,100 x g (Eppendorf 5424) and the cells were resuspended in fresh LM medium. 25 mL LM medium was inoculated with the washed cells, normalizing each culture to a starting OD₆₀₀ = 0.005. The cultures were grown shaking at 30°C. At the desired cell densities, RNA was harvested from three independent cultures using the RNeasy mini kit (Qiagen #74106). RNA levels were normalized to 200 ng/μL and the samples were treated in two sequential reactions with DNase (Turbo DNA-free Kit, Thermo Fisher AM1907). cDNA was generated from 1 μg of RNA using Superscript III Reverse Transcriptase (Thermo Fisher, 18080093) as previously described [19 (link)]. Real-time PCR was performed using a QuantStudio 6 Flex Real-Time PCR detection system (Thermo Fisher) and PerfeCTa SYBR Green FastMix (Quantabio, 95074) as previously described [19 (link)]. In every case, 10 μL reactions were analyzed in quadruplicate technical replicates. Control reactions were performed with samples lacking reverse transcriptase and with samples lacking cDNA templates. Relative transcript levels were measured and normalized to an internal hfq control gene using a comparative ΔΔC_T method. qRT-PCR primers are listed in S2 Table.

Overnight cultures were back-diluted 1:1000 with specified treatments. Subsequently, cultures were grown for an additional 4.5 h prior to 1:10 dilution in water. Samples were heated at 95 °C for 20 min to release viral DNA packaged in phage VP882 virions, linear phage VP882, and host genomic DNA from cells. DNA was further diluted 1:100 in water, and 1 μL was used in qPCR reactions. SYBR Green mix (Quanta) and Applied Biosystems QuantStudio 6 Flex Real-Time PCR detection system (Thermo) were used for real-time PCR. Data were analyzed by a comparative CT method in which the gp69 target gene was normalized to an internal bacterial control gene (hfq).

Excised tissues (leaves and roots) were immediately frozen in liquid nitrogen and stored at −80 °C until gene expression analysis. Total RNA was extracted using a RNAisoTM Plus reagent (TaKaRa, Tokyo, Japan). The obtained RNA sample was treated with RNase-free DNase I, then MMLV-reverse transcriptase (Promega) was used to synthesize the first strand cDNA. Quantitative real time RT-PCR was performed using the SYBR Green PCR Master Mix (TaKaRa, Tokyo, Japan) in the QuantStudio 6 Flex real-time PCR detection system (Thermo Fisher Scientific, Waltham, MA, USA) with appropriate primers. Relative expression of genes compared to ACTIN was calculated using the ΔΔCt method [81 (link)].
The primers used to quantify gene expression were: GmACTIN, 5′-ATCTTGACTGAGCGTGGTTATTCC-3′ and 5′-GCTGGTCCTGGCTGTCTCC-3′; GmPHR25, 5′-AAAGGCCGACAAGAAAGAAACAGG-3′ and 5′-AACCACCGCTACAGCACCAGAAC-3′; GmEXPB2, 5′-TACCCTCCTCTTGTTTCAACCCT-3′ and 5′-AGCACCACCTTCACTACCGTCC-3′; GmYUCCA14, 5′-GATCTTCCATCCTTGGAGGC-3′ and 5-GCAACAGTATGCACTTGACCTTAC-3′; GmTIR1C, 5′-GGTGACAAGGCCCTTTTG-3′ and 5′-CTCACCGAGCAGGAGGAC-3′;

V. cholerae overnight cultures were back-diluted 1:100 into fresh medium and grown to OD₆₀₀ = 2.0. Autoinducers were added at the indicated concentrations at the time of dilution. RNA was harvested from three independent cultures using the RNeasy mini kit (Qiagen, #4104) and cDNA was generated as described [73 (link)] with SuperScript III reverse transcriptase (Invitrogen, #18080–044) using 1.5 μg of RNA. Real-time PCR analyses were performed as described [73 (link)] using a QuantStudio 6 Flex Real-Time PCR detection system (ThermoFisher) and the Sybr Green mix (Quanta, #95074). Quadruplicate technical replicates for each experiment were analyzed by a comparative C_T method (Applied Biosystems) in which the relative amount of hapA [74 (link)] was normalized to the internal hfq control RNA to determine the relative RNA levels. hapA transcript levels in V. cholerae mutants were normalized to WT levels.

Triplicate colonies of WT and ΔahyI Aeromonas sp. ARM81 and V. fischeri strains were each resuspended in 1 mL fresh growth medium and incubated at 30 °C until the cultures reached OD₆₀₀ ~0.5. Cultures were back-diluted 1:100 into a fresh growth medium and combined at a 1:5 ratio of Aeromonas sp. ARM81:V. fischeri. The Aeromonas sp. ARM81 monoculture control was prepared in parallel by dilution of the Aeromonas sp. ARM81 culture 1:5 in the growth medium. The mono- and cocultures were dispensed into a 96-well plate and incubated at 30 °C with shaking for ~10 h, at which point 10-uL aliquots were collected, heated to 95 °C for 10 min, and diluted 1:1,000 in water. SYBR Green mix (Quanta) and the Applied Biosystems QuantStudio 6 Flex Real-Time PCR detection system (Thermo) were used for real-time PCR. Data were processed and analyzed (Pfaffl method) by comparing the relative amplification within samples from reactions using an ARM81ld phage-specific primer pair (targeting cI_ARM81ld, SI Appendix, Table S3) to that from reactions using an Aeromonas sp. ARM81 host-specific primer pair (targeting rpoB, SI Appendix, Table S3). The relative phage ARM81ld viral load was determined by dividing the ARM81 phage-to-host amplification ratio from each coculture condition by that of the Aeromonas sp. ARM81 monoculture.