Lev simplyrna tissue kit

RNA was harvested from cells or tumor homogenates using the Maxwell 16 automated workstation (Promega) and LEV simply RNA Tissue kit (Promega). RNA was then analyzed for concentration by a NanoDrop 2000 (Thermo Fisher) prior to cDNA synthesis using the SensiFAST cDNA synthesis kit (Bioline) with 1 µg of RNA per sample. cDNA and SSO advanced SYBR green universal supermix (BioRad) were then combined with target-specific primers on a CFX96 Touch Real-Time PCR Detection System (BioRad). Oligo sequences for qRT-PCR consisted of: Akap9 forward 5’-TTACCATTGCAGAATAGGTACCCG-3’ reverse 5’-AACGGATTATCTCCTCATGCC-3’; Cdk6 forward 5’-TGGTCAGGTTGTTTGATGTGTGC-3’ reverse 5’-AGTCCAGACCTCGGAGAAGC-3’; Cdk14 forward 5’-TTGTCCGAGAGTTTCAGCCG-3’ reverse 5’-TTGTGACACATATCTCATCAAAGGT-3’; Dbf4 forward 5’-ACGAAGATCTCGAAACTCACC-3’ reverse 5’-AAGAAAGGGACCCGACACTG-3’; Dusp4 forward 5’CATCGAGTACATCGACGCAG-3’ reverse 5’-ATGAAGCTGAAGTTGGGCGA-3’; Fzd1 forward 5’-GAGGTGCACCAGTTCTACCC-3’ reverse 5’-TCACACTTGAGCGTGTCTGG-3’; Gapdh forward 5’-AGGTCGGTGTGAACGGATTTG-3’ reverse 5’-TGTAGACCATGTAGTTGAGGTCA-3’.

Total RNA was extracted from brains and archived at −80 °C for each mouse, using Maxwell® 16 automated equipment for nucleic acid purification with LEV simplyRNA tissue kit (Promega, Sunnyvale, CA). Quantification and quality checking were performed using a Cytation 5 Imaging Multi-Mode Reader (BioTek).

RNA was harvested from cells using the Maxwell 16 automated workstation (Promega) and LEV simplyRNA Tissue Kit (Promega). RNA was then analyzed for concentration by a NanoDrop 2000 (Thermo Fisher Scientific) before cDNA synthesis using SensiFAST cDNA Synthesis Kit (Bioline, Meridian Bioscience, Inc.) with 1 μg of RNA per sample. cDNA and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) were then combined with target-specific primers on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). A heatmap was generated using GraphPad Prism 8. Primers included CXCL1 Forward 5′-AGTCATAGCCACACTCAAGAATGG-3′ reverse 5′-GATGCAGGATTGAGGCAAGC-3′; CXCL2 forward 5′-CTCAAGAATGGGCAGAAAGC-3′ reverse 5′-AAACACATTAGGCGCAATCC-3′; CSF1 forward 5′-CACATGATTGGGAGTGGACA-3′ reverse 5′-TAATTTGGCACGAGGTCTCC-3′; CSF2 forward 5′-CAGCCTCACCAAGCTCAAG-3′ reverse 5′-AATCTGGGTTGCACAGGAAG-3′; mARG1 forward 5′-GTGAAGAACCCACGGTCTGTG-3′ reverse 5′-CTGGTTGTCAGGGGAGTGTTG-3′; mINOS forward 5′-CACCTTGGAGTTCACCCAG-3′ reverse 5′-ACCACTCGTACTTGGGATGC-3′; mS100A8 forward 5′-GGAAATCACCATGCCCTCTA-3′ reverse 5′ -TCCTTGTGGCTGTCTTTGTG-3′; and mNOX2 forward 5′-ACTGCGGAGAGTTTGGAAGAG-3′ reverse 5′-GGTGATGACCACCTTTTGCTG-3′.

We quantified gene expression of GOX (F: GAG GGC GGA AAA TCA TCA GAC C; R: AGG ATT ACC CGA GAT CAC CTG C; [32 (link)]) and Def1 (F: TGC GCT GCT AAC TGT CTC AG; R: AAT GGC ACT TAA CCG AAA CG; [33 (link)]) from the heads of eight individuals per colony for each treatment and time period using only the 14 day old bees. Briefly, total RNA was extracted using the Maxwell® system with the LEV simplyRNA tissue kit (Promega). cDNA synthesis was performed using the QuantiTect Reverse Transcription Kit (Qiagen Inc.) following the manufacturer's protocols. qPCR reactions were conducted using a CFX96™ Real-Time PCR (BioRad, Inc.). Amplification was performed in 10 µl volumes using PowerUP SYBR Green Master Mix (Applied Biosystems) under the following thermal protocol: 95°C hold for 20 s, 40 cycles of 95°C for 1 s, 60°C for 5 s followed by a melt-curve dissociation analysis. All reactions included three technical replicates and qPCR data were expressed as the threshold cycle (C_t) values normalized to expression of β-actin (F: TGC CAA CAC TGT CCT TTC TG; R: AGA ATT GAC CCA CCA ATC CA; [34 (link)]) and calculated using the 2^−ΔΔCt method following standard protocols [35 (link)]. The average for the colony at T₀ (pre-infection) was used as the calibrator, and so normalized expression values were made relative to this.