Cfx real time pcr detection system

Total RNA (isolated as described before) from adult and nymphal (2^nd—3^rd instars) H. vitripennis was used to study the expression pattern of the conserved and novel microRNAs. The miRNA expression was measured using a two-step process. In the first step, a stem-loop (RT) primer (designed based on previous reports [39 (link)]) was hybridized to the miRNA and reverse transcribed in a pulsed RT reaction [40 (link)]. In the second step, the RT reaction product was PCR amplified using a miRNA specific forward primer and a universal reverse primer (S1 Table) in real time with SYBR green chemistry using a Bio-Rad CFX Real-Time PCR detection system [40 (link)]. Quantification of the relative changes in miRNA expression was performed using the method previously described [40 (link)]. In this relative quantification method, the sample reference was chosen as miR10a and the endogenous control was chosen as ubiquitin [41 (link)]. The data for relative quantities were converted to fold differences by logarithmic transformation to express the data as a normal distribution. The data presented are the averages of three measurements.

Protein stability was analyzed with thermo shift assays by measuring the fluorescence of SYPRO Orange (Thermo Fisher Scientific) upon binding the hydrophobic pocket of proteins with increased temperature gradient with manufacturer recommended protocol (Bio-Rad CFX Real Time PCR Detection System, Protein Thermo Shift). Briefly, purified protein (0.1 μg) was mixed with 5× SYPRO Orange using 10% glycerol in TBS to adjust final volume to 25 μL. Temperature gradient was applied as 15–95 °C with 0.5 °C ramp on Bio-Rad CFX96 real-time qPCR machine. Melting temperature was determined by the temperature (X-axis) at the peak of melting peak from CFX Maestro Software (Bio-Rad). Melting peaks were generated as the first derivatives of the melting curve by the CFX Maestro Software (Bio-Rad) during the run. Experiment was repeated with two batches of purified enzymes with six replicates in total. Melting temperatures of WT and mutants were analyzed and graphed in Prism 7. Statistical analysis was performed with One-way ANOVA in Prism 7.

Total RNA was extracted from adipose tissue samples using the Trizol reagent (Invitrogen, USA). The concentration and purity of isolated RNA were evaluated by calculating the ratio of optical density at 260 nm (OD260) and 280 nm (OD 280), and the integrity of RNA was determined by the 18S and 28S ribosomal bands. RNA (2 µg) was used for reverse transcription using the GoScript Reverse Transcription System (Promega, USA). Quantitative real-time PCR analysis was performed with a CFX Real-Time PCR Detection System (Bio-Rad, USA). Each reaction included: 1 µL of cDNA, 0.5 µL of each primer (10 µmol/L), 10 µL of SYBR Premix Ex TaqTM (TAKARA, Japan) and 8 µL of sterile water. The mRNA amplification conditions were 1 min at 95 °C, followed by 44 cycles of 5 s at 95 °C and 30 s at 60 °C, then 0.5 °C increments every 5 s from 55 to 95 °C. All the PCR efficiencies ranged from 90 to 105%.
The primers were designed using Primer Premier 6.0 software (Premier, Canada) with their sites spanning introns. The sequences were as follows SFRP4, forward 5′-GGACCCTGCCAAGTTCAAGA-3′, reverse 5′-ACGGCATACGTGTCGTAGTC-3′; β-actin, forward 5′-AGGTCATCACCATTGGCAAT-3′, reverse 5′-ACTCGTCATACTCCTGCTTG-3′. Threshold cycle values were recorded and relative gene expression was calculated using the formula 2^{−ΔΔ CT}.

For analysis of mRNA expression levels of Ddx19a, cells were harvested 13 days after transduction with the specific shRNAs (Supplementary Table S3) and antibiotic selection. RNA was extracted using the RNeasy Plus mini Kit (QIAGEN). Reverse transcription and quantitative PCR were performed in one step using the Luna® Universal One-Step RT-qPCR Kit (NEB) and a CFX Real-Time PCR detection system (Bio-Rad). Beta-2-Microglobulin was used for normalization. qRT-PCR primers are described in Supplementary Table S5.

qPCR was performed using as templates DNA of T. aestivum, Th. ponticum, Th. Intermedium, and four accessions of partial wheat-wheatgrass hybrids, WWGH 548, WWGH ZP26, WWGH 4044, and WWGH 166.
Primers for selected clusters were designed using Primer3 program (primer length 20 bp, Tm = 60 °C, see Table 2 for primer sequences). For cluster 19-202, two pairs of primers were selected for different repeat regions in order to evaluate their performance (Table 2). The qPCR was carried out using CFX Real-Time PCR Detection System (Bio-Rad) and Real-Time PCR Mix reaction mixture with Eva Green (Syntol Ltd., Moscow, Russia) according to the manufacturer’s protocol. Primers were synthesized at Syntol Ltd. (Moscow, Russia). The reference gene used was VRN1. The primer concentration was 10 ng/μL, and the DNA concentration was 0.4 ng/μL. The amplification program was as follows: pre-incubation for 10 min. at 95 °C, then 40 cycles: denaturation for 10 s at 95 °C; primer annealing for 30 s at 60 °C.