Abi steponeplus cycler

Total RNA used for quantitative RT-PCR (qRT-PCR) analysis was extracted from the different developmental stages of the seeds of the two pea cultivars with three biological replicates using the same method as described above. For qRT-PCR, approximately 3 μg of total RNA was digested using RNase-free DNase I (TaKaRa, Kyoto, Japan) at 37°C to remove any genomic DNA. The digested RNA was then reverse-transcribed to cDNA using MMLV transcriptase (TaKaRa, Kyoto, Japan) in a 20 μl reaction. The cDNAs were then used as templates in qRT-PCR reactions using the SYBR Green Master Mix (TaKaRa, Kyoto, Japan) with gene-specific primers. The reactions were performed on an ABI StepOnePlus™ cycler (Applied Biosystems, USA). The two-step quantitative RT-PCR program began at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 20 s. Fluorescent signals were collected at each polymerization step. Expression was calculated as 2^−ΔΔCt (Livak and Schmittgen, 2001 (link)) and was normalized to that of the control gene β-tubulin (Die et al., 2010 (link)). The primers used for qRT-PCR are listed in Supplementary Table S1. The correlation between log2 normalized qRT-PCR and RPKM values was plotted using Origin 8 (OriginLab, USA).

Total RNA was isolated with the RNeasy Kit (Qiagen, Hilden, Germany) and transcribed into cDNA (High Capacity cDNA Reverse Transcription Kit; Applied Biosystems, Foster City, CA, USA). cDNA was mixed with POWER SYBR Green Master Mix and qPCR performed using an ABI StepOnePlus cycler (Applied Biosystems, Foster City, CA, USA). The housekeeping genes HPRT1, SRP14 and OAZ1 were used for normalization. Primers for realtime analysis are listed in S1 Table. All experiments included two technical and three biological replicates.

Total RNA was isolated from bone tissue and BMSCs using TRIzol Reagent following the manufacturer’s protocol. The total RNA was reverse transcribed using a first strand cDNA synthesis kit. Then, RT-qPCR was performed using a SYBR Green qPCR Master Mix Kit and ABI StepOnePlus cycler (Applied Biosystems, Foster City, CA, USA) with 40 cycles. Relative expression of gene was calculated for each gene by the 2^-ΔΔCT method with glyceraldehyde 3-phosphatedehydrogenasefor normalization. The rat primer sequences for the genes used in this study were shown in Table S1.

Total RNA was isolated with the innuPREP RNA Mini Kit (Jena Analytics) and transcribed into cDNA (High Capacity cDNA Reverse Transcription Kit; Applied Biosystems). cDNA was mixed with POWER SYBR Green Master Mix and qPCR performed using an ABI StepOnePlus cycler (Applied Biosystems). The housekeeping genes GAPDH, RPL32, and OAZ1 were used for normalization. Primer sequences are provided upon request. All experiments included two technical and three biological replicates.

Real-time RT-PCR was performed using hydrolysis probes and the 2x Taqman Universal PCR Mastermix with an ABI Stepone Plus Cycler (Applied Biosystems, Darmstadt, Germany). Reactions were performed in duplicates in a volume of 10 μl with specific primers and probes (SIGLEC7 Hs00255574_m1 (exon spanning probe; PCR efficiency 95%), PPIA Hs99999904_m1 PCR efficiency 100%, β-Actin Hs99999903_m1 (exon spanning probe; PCR efficiency 94%), Tubulin Hs00362387_m1 (exon spanning probe; PCR efficiency 99%), all Applied Biosystems, Darmstadt, Germany). Primer efficiencies were tested in dilution series of a single exemplary investigated blood sample. For all amplicons, a cDNA component control and a control without reverse transcriptase were performed. Cycling program: 20 s 95°C and 50 Cycles: Denaturation 1 s 95°C followed by 20 s 60°C annealing and elongation. Results are presented as C_T or RQ to a calibrator (ΔC_T) [18 (link)] (StepOne Software Version 2.2.2). Real-Time PCR data were efficiency corrected and normalized either to individual reference or to mean reference gene expression [19 (link)].