Superscript 3 rt

Taqman gene expression assays (mouse) were purchased from Life Technologies: Ltβ (Mm00434774_g1), Ccl20 (Mm00444228_m1), Ccl2 (Mm00441242_m1), Cxcl1 (Mm04207460_m1), Cxcl2 (Mm00436450_m1), Csf2 (Mm01290062_m1), Cxcr3 (Mm99999054_s1), Gapdh (Mm99999915_g1), LtβR (Mm00440235_m1),Ltα (Mm00440228_gH). Superscript III RT (Life Technologies) kit was used to synthesize cDNA from RNA that was isolated from pooled dura- and parenchyma-derived 4T1 cancer cell variants. The dCT values for individual probes were normalized to the Gapdh control.

To avoid the nonspecific priming exhibited in reverse transcription of viral RNA, strand‐specific amplification was accomplished in two steps, following the method of Vashist and colleagues (Vashist et al, 2012 (link)). First, viral negative‐strand RNA was reverse transcribed with SuperScriptIII RT (Life Technologies, Cat. #18080‐044) in half‐reactions following manufacturer instructions, with 5 µl of extracted TNA in each 10‐µl RT reaction. A primer containing a non‐viral Tag sequence, Tag‐E_Sarbecco‐F, was used to prime the cDNA synthesis. Second, only cDNA resulting from the tagged RT reaction was amplified in real‐time PCR reactions using QuantiTect Multiplex PCR kits. Each 30 µl PCR contained 5 µl of cDNA, 4.7 µl water, 14.3 µl NoROX (Qiagen, Cat. #204745), 0.7 µl Hi Rox (Qiagen, Cat. #204545), 2.5 µl each of 10 uM Tag‐F and E_Sarbecco‐R primers, and 0.3 µl of 10 µM E_Sarbecco‐P probe. PCR was performed on an ABI 7500 real‐time PCR system with the following cycling parameters: 15′ at 95°C followed by 40 cycles of 60″ at 94°C and 60″ at 60°C (Table 3).

Each single CTC was transferred to 0.2 ml PCR tube and subjected to lysis and RNA extraction according to the manufacturer’s specifications (Single Cell Lysis Kit, Thermo Fisher Scientific, MA, USA). 2.5 μM oligo (dT) primers and 0.5 mM dNTP Mix (all Life Technologies, Singapore) were added into the lysed CTC sample, which was subsequently incubated at 65°C for 5 min and cooled on ice for at least 1 min. 1x first-strand buffer, 5 mM DTT, 10 U RNaseOUT Recombinant RNase Inhibitor, and 50 U SuperScript III RT (all Life Technologies, Singapore) were used, made up to a final volume of 20 μl in nuclease-free water. The final product was incubated at 25°C for 5 min, 55°C for 60 min, and 85°C for 5 min for reverse transcription on a C1000TM Thermal Cycler (Bio-Rad, Hercules, USA).

In total, 1 μl of 50 μM oligo (dT) ₂₀ primers (Life Technologies) and 1 μl of 10 mM dNTP Mix (Life Technologies) were added to 5 μl of each lysed RNA sample. The sample was incubated at 65 °C for 5 min and subsequently cooled on ice for at least 1 min. In all, 1 × first-strand buffer, 5 mM DTT, 40 U RNaseOUT Recombinant RNase Inhibitor, 200 U SuperScript III RT (all Life Technologies) were added to a final volume of 20 μl. The following thermal setting was applied on a Verity 96-well Thermal Cycler (Applied Biosystems): 25 °C for 5 min, 55 °C for 60 min and 85 °C for 5 min. Each RT product was diluted to a final volume of 40 μl to avoid qPCR inhibition.

qRT-PCR was conducted as previously described [3 (link)]. Total RNA used in qRT-PCR was extracted from leaves and roots using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and treated with DNA-free™ reagents (Ambion, Life Technologies, Grand Island, NY, USA) for removing genomic DNA. cDNA synthesis was carried out using SuperScript^® III RT (Life Technologies, Carlsbad, CA, USA) with stem loop primers. Four genes encoding glyceraldehyde 3-Pi dehydrogenase, heat shock protein 70, cyclophilin and α-tubulin, respectively, were used as control genes for normalizing qRT-PCR miRNA expression levels. The RG 6000 Rotor-Gene Real-Time Thermal Cycler (Qiagen, Valencia, CA, USA) was used for performing qRT-PCR assays.

Total RNA was extracted from the cell culture pellet using TRIzol Reagent (Life technologies, UK) and ssRNA was removed by precipitation in 2 M lithium chloride (Sigma, UK) overnight as described (Maan et al., 2007 (link)). The dsRNA (8 μl) was denatured by heating at 95 °C for 5 min and the first cDNA strand was synthesised using SuperScript III RT (Life technologies, UK) and then the second strand was synthesised using NEBNext (New England BioLabs, UK) according to the manufacturers' instructions. Double stranded (ds) cDNA was purified using the Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare, UK) and quantified with the Qubit dsDNA HS Assay kit (Life technologies, UK). The concentration of dscDNA was then adjusted to 0.2 ng/μl with 10 mM Tris-HCl, pH 8.0 buffer. Libraries were prepared using the Nextera XT library preparation kit and sequencing was performed using MiSeq Reagent kit v2 (Illumina, USA) on the MiSeq benchtop sequencer.

Sort plates were thawed on ice and briefly centrifuged before adding 11.7 µl per well of ice-cold RT-PCR master mix containing 5.8 µl nuclease free water, 4.8 µl 5× FS buffer (Life Technologies), 1 µl 25 mM dNTP mix (Roche Applied Science, Indianapolis, Indiana), and 0.1 µl random primers (Life Technologies, catalog no. 48190-011) per well. Plates were again briefly centrifuged, incubated at 65°C for 5 minutes, and returned to ice. Then 8.3 µl of ice-cold RT-PCR master mix (2) containing 4.8 µl nuclease-free water, 0.8 µl 5× FS buffer, 0.2 µl RNasin, 2 µl 0.1M dTT, and 0.5 µl SuperScript III RT (Life Technologies) were added per well, and cDNA was generated with the following PCR program: 1 cycle for 5 minutes at 25°C, 1 cycle for 60 minutes at 50°C, 1 cycle for 15 minutes at 70°C, and 4°C forever. cDNA was stored at 4°C (short-term) or −20°C (long-term).