Abi 7900 real time pcr machine

To examine gene-silencing efficiency of isolated knockdown constructs, real-time qPCR was performed. The total RNAs were isolated from the mid-log phase cell cultures in the SC-U 2% glucose medium of the mutant strains and the control strain using the RNeasy mini kit (Qiagen). The cDNA synthesis was performed by the Transcriptor First Strand cDNA Synthesis kit (Roche, Indianapolis, IN). In each reaction, 5 μg of the total RNAs was used as template, and the gene-specific reverse primers for the target genes and the internal control gene ACT1 were added in the same reaction. For control samples, only the reverse transcriptase was omitted. The primers for the cDNA synthesis were named as ‘gene-name Rev' (Supplementary Table 5). qPCR reaction and data analysis were performed in a Taqman ABI 7900 real-time PCR machine using Applied Biosystems Power SYBR Green PCR Master Mix (Life Technology, Grand Island, NY) following the manufacturer's instructions. Primers for qPCR reactions were named as ‘qPCR gene-name For' and ‘qPCR gene-name Rev' (Supplementary Table 5).

Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was performed to validate microarray results. Four transcripts were selected based on their importance to crowding stress response from this microarray experiment and previous studies (S1 File) [19 , 37 (link)]. Ubiquitin conjugating enzyme was selected as the endogenous control. Invitrogen Superscript First-Strand Synthesis System (Invitrogen) was used to synthesize cDNA from the same total RNA samples. Primer Express Software Version 3.0 (Applied Biosystems, Foster, CA) was used to design primers. Power SYBR Green Master Mix (Applied Biosystems, Foster, CA) and ABI 7900 real time PCR machine were used for performing RT-qPCR. SDS2.4 software (Applied Biosystems, Foster, CA) was used to analyze the RT-qPCR result and identify threshold values. Three technical replications were used for each sample and averaged for the analysis when PCR efficiency was between 90 and 100% and R² close to 0.99 [38 (link)]. The cycle threshold values were normalized to the expression of control genes and the ∆∆Ct method was used for comparing the gene expression values involved in crowding stress [38 (link)].

Total RNAs were extracted using a TRIzol kit according to the user’s manual (Invitrogen, Carlsbad, CA, USA). For each sample, 1 µg of total RNAs was reverse-transcribed as cDNA template using a kit (TaKaRa, Kusatsu, Japan). qRT-PCR was carried out in a mixture of 20 µL consisting of cDNA, gene specific primers (Table S1) and 10 µL 2X mix (TaKaRa), water was added to the mixture to a final of 20 ul. qPCR was performed on an ABI 7900 real-time PCR machine according to the manufacturer’s instruction (Applied Biosystems). For each sample, three biological replicates were performed and cDNA were amplified in triplicate by quantitative PCR. The relative expression values were determined by using rice Ubiquitin gene as reference and the comparative Ct method (2- ΔΔCt).

Sequential stages of IM development were obtained by sampling the IM every 2 days starting from the booting stage (time of IM initiation) for RNA preparation. Total RNAs were prepared using a TRIzol kit according to the user's manual (Invitrogen). For detection of IPA1 transcripts, 1 μg of total RNAs was used for cDNA synthesis with a reverse transcription kit (TaKaRa). qRT-PCR was performed in a 20 μl volume with 2 μl cDNA, 0.5 μM gene specific primers (Supplementary Table 5) and 10 μl 2 × mix (TaKaRa) supplemented to 20 μl by water on an ABI 7900 real-time PCR machine according to the manufacturer's instruction (Applied Biosystems). The rice Actin gene (LOC_Os03g50885) was used as the internal control. Northern blot analysis was carried out to confirm IPA1 transcript levels with the rice Actin probed as loading control. For detection of miR156 and miR529, the same RNA were reverse-transcribed by the miRNA RT-PCR Kit (TaKaRa) and qRT-PCR was performed following the same procedure described above, and universal adaptor primers (supplied by the Kit) and Uni-miRNA primers were used with 5 s rRNA as internal control (Supplementary Table 5).

Tissues were mechanically homogenized using a bead basher, and RNA was extracted using the TRIzol method according to the manufacturer's instructions (Ambion, Austin, TX). cDNA was generated using High-Capacity cDNA reverse transcription kits according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Quantitative PCR was performed using the SYBR FAST master mix (Kapa Biosystems, Woburn, MA) and SYBR green-optimized primer sets run on an ABI 7900 real-time PCR machine (Applied Biosystems). Threshold cycle (C_T) values of all genes measured in each sample were normalized relative to beta-actin (Actb) gene expression. Primer sets are listed in Table 1.

Total cellular RNA was isolated from samples taken in TRIzol reagent (Ambion) per the manufacturer’s protocol using 1-bromo-3-chloropropane (BCP) and isopropanol. For qRT-PCR, 10 µg of RNA per sample was reverse transcribed into cDNA using random hexamer primers and semiquantitative PCR was performed using the Sybr green method on an ABI 7900 real-time PCR machine (Applied Biosystems) using gene-specific primers. See Table S1 for a complete list of primer sequences. Threshold cycle (C_T) values were normalized to 18S rRNA and compared using the ΔΔC_T method (86 (link)). For NanoString, 100 ng RNA from each sample was subjected to fluorescent probe hybridization (see Table S2 for complete list of gene targets and probe sequences) and fluorescent bar codes corresponding to individual target mRNA molecules were counted automatically with an nCounter analysis system (NanoString Technologies). All mRNA quantification steps were performed by the Genomics Research Core at the University of Pittsburgh. Raw mRNA counts for each target gene underwent background subtraction and housekeeping gene normalization using nSolver 2.6 software (NanoString Technologies).