Abi prism 7000 system

Total C. albicans SC5314 RNA (1 μg) from yeast and hyphae was reverse-transcribed into cDNA using the iScript^TM cDNA synthesis kit (Cat# 170–8890, Bio-Rad, Hercules, CA) and amplified by iTaq^TMFastSYBR Green Supermix with ROX (Cat#172–5103, Bio-Rad, Hercules, CA) using specific primer sets for each target gene and the housekeeping gene PMA1. Fluorescence from DNA-SYBR Green complex was monitored by the ABI PRISM 7000 System (Applied Biosystems, Foster City, CA) throughout the PCR reaction. The level of target mRNA relative to the mean of PMA1 was calculated by the comparative Ct method. ALS3 mRNA was detected with primers ALS3-F and ALS3-R.
To detect GFP mRNA in strain SKD233 (HCRc), total RNA from cells grown under hyphal conditions was used to prepare first strand cDNA using primer GFPstop that hybridizes to the 3' end of GFP mRNA. The first PCR, using nested primer GFP109 and the abridged anchor primer (AAP) (ThermoFisher Scientific, Waltham, MA) revealed only the hypha-specific GFP transcript under hyphal growth conditions. The second PCR using GFP109 and the abridged universal amplification primer (AUAP) to amplify products from the first PCR revealed a product that was specific to yeast growth conditions and labeled HCR-Y. For strains with terminator or terminator control sequences, GFP mRNA was detected with primers GFP70-F and GFP70-R.

N. tabacum ‘K326’ seedlings were raised in a growth chamber at 22 °C with a 12/12 h light-dark photoperiod. For the tissue-specific expression analysis, cotyledons were sampled from one-week-old seedlings, and the leaf, root, stem epidermis, and the stem with its epidermis removed were sampled from three-week-old seedlings.
Total RNA was extracted and removed the residual DNA using DNase I. Quantitative real-time PCR (qRT-PCR) and semi-quantitative RT-PCR were employed to determine the relative mRNA transcriptions of HD-ZIP IVs in five tobacco tissues using the gene-specific primers (Additional file 1: Table S1). L25 gene was selected as an internal control. q-PCR reaction was performed on an ABI PRISM 7000 system (Applied Biosystems, USA) with the SYBR Green RT-PCR Kit (Takara, China). Each reaction was run in triplicate, and analysis was performed using the 2^-ΔΔCT method [50 (link)].

RNA was isolated from renal cortical tissues of rats by the phenol–chloroform extraction method and cDNA prepared as described previously [26 (link)]. The mRNA expression of α-smooth muscle actin (Sma) and fibroblast growth factor (Fgf) 23 was analyzed by real-time PCR using an ABI Prism 7000 system with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The oligonucleotide primers for rats used were as follows (forward and reverse, respectively): 5′-ACGGCGGCTTCGTCTT CT-3′ and 5′-CCAGCTGACTCCATGCCAAT-3′ for α-Sma [27 (link)]; 5′-TTGGATCGTATC ACTTCAGC-3′ and 5′-TGCTTCGGTGACAGGTAG-3′ for Fgf23 [28 (link)]; 5′-CCCTG GCTCCTAGCACCAT-3′, and 5′-CCTGCTTGCTGATCCACAT CT-3′ for β-Actin [27 (link)]. The abundance of target genes were normalized to β-Actin (as internal control) and analyzed by the 2^−ΔΔCt method as described previously [29 (link)].

Twelve LysM domain-containing protein genes are encoded by B. bassiana [30 (link)]. To examine the expression of these genes, total RNA was extracted from the mycelia or blastospores harvested from SDB and the conidial spores or hyphae from the PDA plates. To determine gene expression during fungal in vivo infection, the last instar larvae of wax moth (G. mellonella) were individually injected with 10 μl of spore suspension (10⁷ spores/ml) for 60 hrs. Insect hemolymph was collected on ice, and fungal hyphal bodies were harvested by gradient centrifugation using Centricoll (Sigma-Aldrich) [30 (link)]. Each RNA sample was converted to cDNA using an AffinityScript multiple-temperature cDNA synthesis kit (Toyobo). qRT-PCR analysis was performed using a SYBR Premix Ex Taq kit (Takara) containing the primer pairs for different genes (S3 Table) on an ABI Prism 7000 system (Applied Biosystems). The β-tubulin gene (BBA_07018) of B. bassiana was amplified as an internal control. To determine insect antifungal gallerimycin gene expression, the last instar wax moth larvae were individually injected with the spore suspensions of WT and mutants for 36 hrs. Insect fat bodies were then dissected on ice and collected for RNA extraction to quantify the expression of the antifungal gene [9 (link)].