Mx3000p real time pcr instrument

Total RNA was extracted using an RNA prep pure Plant Kit (TANGEN) and first-strand cDNA was synthesized using a PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa) according to the manufacturer’s instructions. Quantitative RT-PCR was performed following the instructions of the SYBR^® Premix Ex Taq™ Kit (TaKaRa) on a Mx3000P real-time PCR instrument (Agilent). The experiments were performed with three biological replicates, each with three plants. Relative gene expression was calculated using the 2^−△△Ct method [51 (link)]. The CsBPC specific primers used for expression pattern analysis in different tissues and under abiotic stress and hormone treatments are shown in Additional file 2. S1.

Total RNAs were extracted from lung tissues using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). By using the template of cDNAs that reversely transcribed by FastKing First-strand cDNA Synthesis Mix (Tiangen, Beijing, China), qRT-PCR was performed on MX3000P Real-Time PCR instrument (Agilent, Santa Clara, CA, USA). The qRT-PCR program was an initial 95°C for 3 min and 40 cycles of 95°C for 15 s and 62°C for 40 s. The relative expression of RKIP was calculated by the 2^-∆∆Ct method. GAPDH was used as the internal control. The primers used in qRT-PCR are listed in Table 1.

Mycobacterium avium subspecies paratuberculosis genomic DNA was quantified in both the undiluted and the diluted DNA extracts following the HT-J qPCR method (Plain et al., 2014 (link)) which targets the IS900 sequence of MAP. Briefly, the reaction mixtures comprised of 5 μl template DNA, 250 nM final concentration of each forward [MP10-1, (5′-ATGCGCCACGACTTGCAGCCT-3′)] and reverse [MP11-1, (5′-GGCACGGCTCTTGTTGTAGTCG-3′)] primers (Kawaji et al., 2007 (link)) and 12.5 μl of SensiMix SYBR Low-ROX qPCR mastermix (Bioline) in a final volume of 25 μl, with amplification performed using an Mx3000P real-time PCR instrument (Stratagene, Agilent). Quantification of MAP DNA was done with reference to the standard curve included in every experiment of 10-fold serially diluted MAP genomic DNA ranging from 10 to 0.001 pg/reaction. The detailed acceptance criteria for HT-J positive qPCR results was previously defined and included a cut-point of ≥0.001 pg target DNA quantity per reaction (Plain et al., 2014 (link)) equivalent to Ct value of 35.1 ± 1.04 (Figure 2C).
Each undiluted DNA extract was tested in a single reaction while diluted DNA extracts were tested in duplicate and a positive mean DNA test result for both replicates was considered a positive sample test result.

Total RNA was isolated by TRIzol reagent (Invitrogen) according to the manufacturer's introduction. Total RNA (2 μg) was used to synthesize cDNA using oligo-dT primers and SSRIII Transcriptase (Invitrogen) for reverse transcription. qRT-PCR was performed using the SYBR Green Supermix enzyme (Agilent Technologies, CA, USA) as we previously described [42 (link)]. Reactions were cycled using an Mx3000P real time PCR instrument (Agilent Technologies) with universal cycling conditions. Relative gene expression quantitation was determined with endogenous reference GAPDH gene and was analyzed using the comparative C_T method [43 (link)]. The quantitative PCR primers were listed in Supplementary Table 2.

Thermal stability was evaluated by nanoDSF using the Tycho NT.6 system (NanoTemper Technologies GmbH, München, Germany)^{67 (link)}. Also, Thermal stability at a constant 37 °C was measured by a thermal shift assay using a Mx3000p real-time PCR instrument (Agilent technologies, Santa Clara, California, USA) and SYPRO orange (Thermo Fisher Scientific)^{68 (link)}.

The RNA of cucumber leaves was isolated following the instructions of the RNA prep pure Plant Kit (TANGEN) and first-strand cDNA was synthesized using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). Quantitative RT-PCR of 10 ABA signaling related genes were performed according to instructions of the SYBR® Premix Ex Taq™ Kit (TaKaRa) using a Mx3000P real-time PCR instrument (Agilent). Each sample was performed in triplicate. Relative gene expression was analysed according to the 2^−△△Ct method [59 (link)]. Primers for the quantitative realtime PCR are listed in Table S3 (see online supplementary material). Gene screening method: All homologous protein sequences of the targer gene were obtained from TAIR (http://www.arabidopsis.org), and then used the obtained sequences to perform BLASTP in the Cucumber Genome Database (http://cucurbitgenomics.org/organism/2) to search for all homologous genes in cucumber. Finally, a phylogenetic tree was constructed with all downloaded protein sequences from Arabidopsis and Cucumber Genome Database, and cucumber gene with the highest homology to the target gene in Arabidopsis was identified as the candidate gene.

Comparative real-time RT-PCR via probe-based detection was performed using cDNA derived from ERS1⁺ and ers1-Δ cells as described above. Primers were designed using Beacon Designer version 7.01 (Premier Biosoft). Each reaction consisted of transcript-specific primer pairs and a FAM-labeled primer probe (Eurofins MWG Operon, Huntsville, AL, USA;Table S3), template cDNA, and Thermo Scientific Absolute Blue qPCR master mix (Thermo Fisher Scientific, Waltham, MA, USA), as per the manufacturer's guidelines, with at least three biological replicates and three technical replicates per plate along with no reverse transcriptase and no template controls. Thermal cycling and fluorescence detection was performed via a Stratagene Mx3000p real-time PCR instrument (Agilent Technologies-Stratagene, La Jolla, CA, USA). Following normalization of Ct values to the ACT1 housekeeping transcript, target transcript changes were deemed significant via Student's t-test if P<0.05 and the fold-change was greater than ±2.0.