Revertaid first strand cdna kit

MDA-MB-231 breast cancer cells were cultured for 24 hours prior to CM treatments. The cells were then treated with CMs from NF-, CAF-, MDA-MB-231- educated monocytes or from control monocytes for 48 hours. At the end of this period, RNA isolation from the cells was performed according to the Zymo Quick-RNA Miniprep Kit protocol. Complementary DNA was synthesized with Thermo Scientific RevertAid First Strand cDNA Kit according to the manufacturer’s instructions. Real-time qPCR was performed using LightCycler FastStart DNA Master SYBR Green (Roche, Basel, Switzerland). The qPCR data were analysed using the Livak model (2^−ΔΔCt). Snail (Snail_reverse 5′-CTGCTGGAAGGTAAACTCTGGA-3′, Snail_forward 5′-CGAGTGGTTCTTCTGCGCTA-3′), Slug (Slug_reverse 5′-TTCTCCCCCGTGTGAGTTCTAA-3′, Slug_forward 5′-CACTGCGATGCCCAGTCTA-3′), and Twist (Twist_reverse 5′-CCCACGCCCTGTTTCTTTGA-3′, Twist_forward 5′-GCCGGAGACCTAGATGTCATT-3′) gene expressions were evaluated. β -actin was used as the reference gene.

The circular RT-PCR (cRT-PCR) protocol for the poly(A) analyses was adapted from Rosenthal et al. (2018) (link). Five micrograms of total RNA had the 5′ cap removed using RppH (New England Biolabs, Ipswich, USA) in a reaction containing NEB Thermopol buffer (New England Biolabs, Ipswich, USA) and RNAseOut (Thermo Fisher Scientific, Waltham, USA) for 1 h at 37°C. To stop the de-capping reaction, RQ1 DNAse stop solution (Promega, Madison, USA) was added and samples were heated for 5 min at 65°C. RNA was purified with the RNeasy^® Mini Kit (Qiagen, Hilden, Germany) before circularization, which was performed using T4 RNA ligase 1 (New England Biolabs, Ipswich, USA), 10% PEG 8000, 50 μM ATP and RNAseOut (Thermo Fisher Scientific, Waltham, USA). cDNA was synthesized using the RevertAid first strand cDNA kit (Thermo Fisher Scientific, Madison, USA) with a specific primer located near the 5′end of the GFP. The region containing the poly(A) was PCR amplified using nested primers (30 cycles reaction) and cloned into pGem^®-T Easy (Promega, Madison, USA). Individual clones were sequenced by Sanger. Sequencing results are provided in Supplementary Material (Data sheet S3 and S4). Annotation of the poly(A) site considered the first nucleotide of the terminator as position “1.” Sequence of all primers used in this experiment can be found in Supplementary Table 1.

Lungs were subjected to total RNA extraction using RNAeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Total RNA concentration was measured using Nanodrop. Then, the RT of total RNA (200 ng) was performed using the Revert Aid First Strand cDNA Kit (Thermo Fisher Scientific) using random hexamers according to the manufacturers’ instructions and stored at −20 °C.
Quantitative real-time PCR (qRT-PCR) was performed using a Maxima SYBR Green qPCR Master Kit (2x) (Thermo Fisher Scientific). Sequences of selected cytokine primers are listed in Table 1. Melting curves were set post the end of the last PCR cycle. The analysis of relative expression was performed using β-Actin as the housekeeping control gene using the 2^(−ΔΔCT) equation.
ΔΔCT of vaccinated groups (at 6 wpv) = ΔCT (vaccination, challenge infection) − ΔCT (vaccination), where ΔCT (vaccination) = CT_V (target gene) − CT_V (B-Actin), and ΔCT (vaccination, challenge infection) = CT_VI (target gene) − CT_VI (B-Actin).
ΔΔCT of control infection groups = ΔCT (Control infection) − ΔCT (Control non-infected/non-vaccinated) [49 (link),50 ]. Viral RNA shedding was also measured in the lungs by qRT-PCR to calculate RNA copy numbers.

For the gene expression analysis, fungal biomass corresponding to the four stages of the life cycle, namely, (i) germlings, (ii) trophic hyphae, (iii) aerial hyphae, and (iv) conidiophores and conidia, were collected from cellophane-covered agar plates or liquid cultures. Total RNA was extracted using an RNeasy Plant MiniKit (Qiagen, Germany) according to the manufacturer’s protocol. cDNA was synthesized with a RevertAid First Strand cDNA Kit (Thermo Fischer Scientific, USA) using the oligo (dT)¹⁸ primer. qPCR was performed using qTOWER (Jena Analytics, Germany) for the genes of interest (listed in Table 1) and calculated by the 2^-ΔΔCt method [75 (link)] using tef1 as the housekeeping gene [76 (link)]. PCR was prepared in a total volume of 20 μl containing 10 μl of iQ SYBR Green Supermix (Bio-Rad, USA), 0.5 μM each primer and 100 ng of cDNA. The program was 40 cycles with 30 s at 95°C and 60 s at 60°C, which was initially denatured for 6 min at 95°C. A melting curve from 55°C to 95°C was generated for each qPCR run.

Cacao samples were macerated in liquid nitrogen until a fine powder was obtained. Total RNA was extracted from 100–150 mg of macerated tissue using the RNAqueous® Total RNA isolation kit according to the manufacturer’s instructions (Thermo Scientific) with modifications as previously described [47 (link)]. Briefly, after the addition of the lysis buffer to the macerated samples, a sonication step was added (10 s pulse/min, 70% output; Gex Ultrasonic processor 130, 130 W) to break polysaccharides which are present in high levels in cacao tissues. This step was conducted on ice. RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific) and its integrity was checked by 1% agarose gel electrophoresis. RNA was treated by DNAse I RNase-free according to the manufacturer’s instructions (Invitrogen). The cDNA was synthesized from 200 ng of RNA using the RevertAid First Strand cDNA kit according to the manufacturer’s instruction (Thermo Scientific). The cDNA quantification was carried out in the same NanoDrop 2000 spectrophotometer.

To analyze the effect of the variants on RNA expression, cDNA was analyzed by restriction enzyme digestion and deep sequencing. First, RNA was extracted according to the manufacturer’s protocol from venous blood collected into PAXgene Blood RNA tubes (Qiagen N.V., Venlo, The Netherlands) from individuals II.1, II.2 and III.2 (Figure 1, family 1). cDNA was synthesized using the RevertAid First Strand cDNA kit (Thermo Scientific Inc., Waltham, MA, USA), with random hexamer primers according to the manufacturer’s instructions. Primers were designed for all exons and intron two to screen for truncations and intron retention (Supplementary Table 1). cDNA was amplified and products were electrophoresed on agarose gels and DNA was visualized by staining with ethidium bromide (EtBr).

Total RNA was extracted using a total RNA extraction kit (Omega, Norcross, GA, USA), according to the manufacturer’s instructions. cDNAs were synthesized using the RevertAid First Strand cDNA kit (Thermo Fisher Scientific, Waltham, MA, USA). The miR-27a expression levels were assessed with qRT-PCR using an ABI 7500 thermal cycler and a High-Specificity miR-27a qRT-PCR Detection Kit (Stratagene Corp, La Jolla, CA, USA), according to the manufacturer’s instructions. U6 small nuclear RNA (snRNA) was used as the internal reference for normalization of the miR-27a levels. The results were calculated and measured using the 2^−∆Ct method.
The expression levels of mRNAs including PPARγ, GREM1, Runx2, Bmp-2, and C/EBPα were verified by qRT-PCR using SYBR Green I (Takara, Dalian, China), according to the manufacturer’s instructions. β-actin was used as the internal reference to normalize the expression levels. Each experiment was performed in triplicate.