Omniscript reverse transcriptase kit

Total RNA was extracted with Qiazol and RNeasy Mini kit (Qiagen, Maryland, USA). DNA was removed using DNase (Qiagen). RNA was converted to cDNA using Omniscript Reverse Transcriptase kit and Oligo(dT)_12-16 kit (Qiagen). Predesigned primers sequences were obtained from PrimerBank (Table 1). qPCR was performed with SYBR green Mastermix (Applied Biosystems) on 7900HT Fast Real-Time PCR system (Applied Biosystems) using the amplification protocol: 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Dissociation curve analysis was also applied to confirm specific amplification. Expression levels were normalized to the house-keeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT1) and results were analyzed as relative expression to HPRT1. Where results involve leptin treatment, they are presented as fold-change compared to untreated cells. Each experiment included at least 3 biological replicates, and results shown are representative of at least 3 independent experiments with consistent outcomes. In some cases, biological replicates from independent experiments were combined for statistical analysis.

Total RNAs were extracted and purified using TRIzol® Reagent according to manufacturer’s (Invitrogen, Carlsbad, CA, USA) instructions and reverse-transcribed using an Omniscript™ Reverse Transcriptase kit (Qiagen, Valencia, CA, USA) with oligo dT primers. “Hot start” RT-PCR was carried out using a Taq PCR Master Mix kit according to manufacturer’s (Qiagen) instructions. RT-PCR was carried out on a MyiQ™ Single-Color Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) with SYBR® Green Supermix (Bio-Rad Laboratories). Relative levels of expression in each assay were obtained by normalizing the Ct values of the tested genes against that of Tubulin. The primer sequences used for mRNA expression are listed in Supplementary Table 1.

Total mRNA was isolated with RNeasy Mini Kit (Qiagen, Venlo, Netherlands, #74104), following the manufacturer's instructions. One microgram was used to synthetize cDNA (20 μl reaction volume) using the Omniscript reverse transcriptase kit and oligo-dt primers (Qiagen, # 205111 and #79237) in a Thermo Hybaid PCR Express (Fischer Scientific, Schwerte, Germany). Real-time PCR analyses of 1 μl of the cDNA products were performed in duplicate with iQ SYBR Green Supermix (Bio Rad, Munich, Germany, #170-8882) in a CFX96 Real time system Thermal cycler (Bio Rad). The primer pairs used for real-time amplification of aSyn are described in Rhinn et al.^{59 (link)} Primers for HPRT^{41 (link)} and QuantiTect Primer assay GAPDH mix (Qiagen #QT00079247) were used for normalization. Relative expression levels were calculated with subsequent ΔCT values that were assessed using the comparative CT method with subsequent ΔCT values normalized to the housekeeping genes HPRT and GAPDH.

RNA was extracted from the fat bodies of six female mosquitoes using the TRIzol method (Invitrogen) according to the manufacturer’s protocol. It was concentrated using the RNeasy MiniElute cleanup kit (Qiagen) for further processing. RNA was treated with DNase I (Invitrogen), after which cDNAs were synthesized from 2 μg of this total RNA using the Omniscript Reverse Transcriptase kit (Qiagen). PCR was performed using the Platinum High Fidelity Supermix (Invitrogen). qRT-PCR was performed using the iCycler iQ system (Bio-Rad) and an IQ SYBR Green Supermix (Bio-Rad). Quantitative measurements were performed in triplicate and normalized to the internal control of actin mRNA for each sample. Real-time data were collected and exported to Excel (Microsoft) for analysis. RNA extraction, cDNA synthesis and subsequent qRT-PCR analysis were done as previously described [15 (link)]. All the primers used are listed in S3 Table.

Fibrin constructs (hMSCs derived from three patients each with three biological replicates) were prepared as aforementioned and cultured with the appropriate differentiation media for a period of up to 14 days (as required) and isolated by trypsin digestion to yield a suspension of cells. RNA extraction was performed using a RNeasy® Micro kit (Qiagen Ltd., UK. Cat. No 74004) and followed the as supplied instructions. RNA concentration was then determined using a P-330 nanophotometer (Implen GmbH, Germany) prior to cDNA synthesis using an Omniscript® reverse transcriptase kit (Qiagen Ltd., UK. Cat. No. 205111) following the as supplied instructions and 50 ng of template RNA. Quantitative reverse transcriptase PCR (RT-qPCR) (with two technical duplicates) was then performed on a StepOne Plus RT-qPCR instrument (Applied Biosystems Inc., USA) with TaqMan® Universal Master Mix (II) (ThermoFisher Scientific Ltd. Cat. No. 4440043) and corresponding TaqMan® gene expression assays for SOX9 (UniGene ID; Hs.647409), PPAR-γ (UniGene ID; Hs.162646), RUNX2 (UniGene ID; Hs.535845) and (housekeeping) GAPDH (UniGene ID; Hs.544577). The raw C_t values were collated and the ΔC_t values determined and exponentiated to give the relative expression levels reported.

Mice were sacrificed at day 7 post irradiation and total RNA of the tumors isolated by TRIzol (Invitrogen) was reverse transcripted into cDNA by Omniscript reverse transcriptase kit (Qiagen, Düsseldorf, NRW, Germany). The quantitative PCR performed by the LightCycler^® 480 SYBR Green I Master reagent (Roche, Basel, Switzerland) was analyzed by CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The mRNA expression of each gene was normalized for the housekeeping gene (β-actin) and the fold change of gene expression in each group was determined by the difference (ΔΔCt value) compared to the control group. The primers of genes are shown in Table 1.