Icycler iq5 multicolor real time pcr detection system

Total RNAs were extracted using the PureLink^TM Plant RNA Reagent (Invitrogen) from fibers (5, 10, 15, and 20 DPA). cDNAs were synthesized from 2 μg of total RNA using the TaKaRa RNA PCR Kit. Quantitative RT-PCR was performed on an iCycler iQ5 Multicolor real-time PCR detection system (Bio-Rad) using the Power SYBR Green PCR MasterMix (Applied Biosystems). Each PCR reaction (10 μL) contained 5 μL of real-time PCR Mix, 0.1 μL of each primer and the appropriately diluted cDNA. The thermal cycling conditions were 95°C for 30 s followed by 40 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 15 s. The GhUBQ10 transcript was used as an internal reference for all the qRT-PCR analyses (Walford et al., 2011 (link)). Each sample was analyzed using three biological replicates and three technical replicates for each biological replicate. The relative gene expression levels were calculated according to the 2^-ΔΔCT method of the system. The primers used are described in Supplementary Table S8.

The melting temperatures of the MhGgH variants were determined using a thermal shift (Thermofluor) assay. Each protein sample (0.5 mg ml⁻¹ final concentration) was centrifuged at 13 000g and 4°C for 15 min, mixed with 5× SYPRO Orange (Life Technologies) in storage buffer and loaded into white 96-well PCR plates (Bio-Rad) sealed with Optical Quality Sealing Tape (Bio-Rad). The plate was heated from 25 to 95°C in 0.5°C steps with 30 s hold time per step on an iCycler iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad) and the fluorescence was followed using a Cy3 dye filter (545 nm excitation/585 nm emission). Each experiment was performed in triplicate. The melting curves were analysed using the CFX Manager software (Bio-Rad) and the melting temperature was determined as the inflection point of the melting curve.

qRT-PCR was used to detect IGF2R and HCN1 mRNA expression levels and the corresponding primers are presented in S18 Table. qRT-PCR was performed using the SYBR PrimeScript RT-PCR Kit (TaKaRa, Dalian, China) with SYBR Green dye as described previously [60 (link)]. Briefly, qRT-PCR reactions were carried out with an iCycler IQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, USA). The qRT-PCR reaction volume was 25 μL, contained 1 μL of cDNA template, 12.5 μL of SYBR Premix ExTaq, 9.5 μL of sterile water, and 1 μL of each gene-specific primer. Thermal cycling parameters were 1 cycle at 95°C for 2 min, 40 cycles of 95°C for 15 s, and 60°C for 34 s. Dissociation curve analysis was done after each real-time reaction to ensure that there was only one product. The qRT-PCR analysis of each sample was done in triplicate.

The SYBR PrimeScript RT-PCR Kit (TaKaRa, Dalian, China) and a reference gene (β-actin) were used for detecting the expression of MAP2K1 (a of the target of novel-mir-8) and PPARα (a of the target of novel-mir-14). The qRT-PCR reactions were carried out with an iCycler IQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, USA). The qRT-PCR reaction contained 1 µL of cDNA template, 12.5 µL of SYBR Premix ExTaq, 9.5 µL of sterile water, and 1 µL of each gene-specific primer (Table 6). Thermal cycling parameters were 1 cycle at 95°C for 2 min, 40 cycles of 95°C for 15 s, 60°C for 34 s. Dissociation curve analysis was done after each real time reaction to ensure that there was only one product. qRT-PCR analysis of each sample was repeated for three times. The quantification of each gene relative to β-actin gene was calculated using the equation: N = 2^−ΔΔCt.

A549 cells grown in 12-well plates were harvested and total RNA extracted using RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. mRNA levels were quantified by two-step real-time PCR. Briefly, 2.5 µg of total RNA per sample was incubated with 3 µg of random primers (Invitrogen Corp., Carslbad, CA, USA) at 70°C for 2 minutes. The reaction was chilled and incubated with reverse transcriptase Superscript II (Invitrogen Corp.) and 1.25 mM dNTP (Applied Biosystems, Foster City, CA, USA) at 42°C for 30 minutes and a third incubation at 70°C. Equal amounts of cDNA were used for the real-time PCR. All reagents, primers and probes are validated Taqman expression assays purchased from Applied Biosystems (RANTES CCL5: Assay ID Hs00174575_m1 cat# 4453320, spans exons 2and 3 with an amplicon length of 63 bp; IP10 CXCL10: Assay ID Hs00171042_m1 cat # 4331182, spans exon 1–2 amplicon length 98 bp; MCP-1 CCL2: Assay ID Hs00234140_m1 Cat. # 433118, spans exon 1–2 amplicon length 99 bp; GAPDH: Assay ID Hs01922876_u1Cat. # 4331182, spans exon 7-7 amplicon length 139 bp). All reactions were performed and analyzed on an iCycler iQ5 multicolor real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). Levels of RANTES, IP-10 and MCP-1 in each sample were normalized and expressed as fold change over GAPDH.

Cells were lysed using a 4 M guanidinium thiocyanate solution and RNA extracted using the RNeasy Mini Kit (Qiagen GmbH, Germany) according to the protocol provided. A total of 1 µg RNA was transcribed into cDNA using oligo-dT-nucleotides (Life Technologies, USA) as primers and M-MLV reverse transcriptase (Promega). Quantitative real-time PCR (qPCR) was performed using the MESA Green qPCR MastermixPlus for SYBR^® ASSAY (Eurogentec) on an iCycler iQ5 multicolor real-time PCR detection system (Bio-Rad) using the following program: initial denaturation for 10 min at 95°C, then 45 cycles of denaturation (15 sec, 95°C), and elongation (60 sec, 60°C). Gene expression was analyzed using the 2^-∆∆CT (cycle threshold) method with ribosomal protein S16 (rP) serving as a reference gene. Please refer to Table 1 for respective primers.

To measure IFNβ mRNA level in virus infected cells, RNA was extracted from cells using the RNeasy mini kit. 500 ng of extracted RNA was used for reverse transcription using oligo (dT)₂₀ primer and SuperScript III reverse transcriptase to generate cDNA. The cDNA was then combined with the primer set and SYBR green super mix. Quantitative real-time PCR (qPCR) reaction was performed in an iCycler IQ5 multicolor real-time PCR detection system (Bio-Rad, Hercules, CA) using the following primers: porcine IFNβ forward, 5′-CCGAATTCGCTAACAAGTGCATCCTCC-3′; porcine IFNβ reverse, 5′-GCGAAGCTTTCAGTTCCGGAGGTAATC-3′; porcine RPL19 forward, 5′-AACTCCCGTCAGCAGATCC-3′; porcine RPL19 reverse, 5′-AGTACCCTTCCGCTTACCG-3′. RPL19 gene transcription was used for normalization of IFNβ expression. Data are presented as relative gene expression to that of mock infected cells using the formula 2^-ΔΔCt (Livak and Schmittgen, 2001 (link)). Real-time PCR was conducted in triplicate for each sample and the mean value was calculated. Final figures represent the results from three independent experiments.