Rnaqueous micro total rna isolation kit

To inhibit Pfn2 expression in vivo, we employed lentiviral delivery of shRNA targeting Pfn2 (sense sequence 5′-AGAAGTGTTCTGTGATCAG-3′) to the IPN or VTA. Lenti-pGIPZ-Pfn2-shRNA-tGFP (2.37 × 10⁸ TU/ml, V3LMM_494369, Dharmacon) or a control virus expressing a non-silencing shRNA (lenti-pGIPZ-scramble-tGFP, 9.06 × 10⁸ TU/ml, RHS4348, Dharmacon) were injected into IPN or VTA and expressed for 4 weeks prior to all experiments. Stereotaxic injections were performed under aseptic conditions using mice aged 6 weeks as described by Molas et al.^{34 (link)}. Virus was injected in the IPN or VTA (unilateral) at coordinates measured from Bregma (in mm, anterioposterior, mediolateral, dorsoventral at 0°): IPN (−3.47, ±0, −4.77), VTA (−3.45, ±0.5, −4.2) using a 26 s gauge 10-μl syringe (701RN, Hamilton) to deliver 300 nl of virus at a controlled rate of 60 nl/min.
Fresh frozen brains were cryosectioned (12-μm), fixed in ice-cold acetone, and dehydrated in ethanol (70%, 90%, 100%) and xylenes. Areas expressing tGFP were isolated by laser capture microdissection (LCM; Arcturus). RNA was isolated using RNAqueous-Micro Total RNA Isolation Kit (Ambion) for RT-qPCR.

Total RNA from the laser-captured bronchiolar epithelial cells was extracted using an RNAqueous-Micro Total RNA Isolation kit (Ambion, Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. The quantity and quality of the RNA extract were assessed by microcapillary electrophoresis with a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) using an Agilent RNA 6000 Pico kit (Agilent Technologies, Santa Clara, CA, USA). Total RNA was reverse transcribed for complementary DNA synthesis (cDNA) using a High-Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions.

A total of 10⁸ logarithmic-phase promastigotes were pelleted for RNA extraction using the RNAqueous-Micro total RNA isolation kit (Ambion). RNA quantification prior to cDNA synthesis was done using Qubit and the Qubit RNA BR assay (Life Technologies, Inc.). Synthesis of cDNA was performed using Transcriptor reverse transcriptase (Roche) according to the manufacturer’s instructions. qPCRs were run on a LightCycler 480 (Roche) with a SensiMix SYBR No-ROX kit (Bioline) as the SYBR green source; the primers used are displayed in Table S2. Expression levels were assessed with qBase+ (Biogazelle) using for normalization two genes previously shown to be very stable: SAT (LdBPK_340035000) (40 (link)) and LdBPK_240021200 (25 (link)). The expression of both normalization targets was confirmed by qBase+ as stable during the course of the experiment. Data were then rescaled to the BPK026 WT. An increase of 1.5-fold compared to the BPK026 WT was considered significant.

RNA was extracted from BLs using an RNAqueous™-Micro Total RNA Isolation Kit (Ambion, Austin, TX, USA), according to the manufacturer’s instructions. The concentration of extracted total RNA was quantified by a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and presented in Table S1. Using the RNA, complementary DNA (cDNA) was synthesized by a Maxime RT premix kit (iNtRON, Gyeonggi, Korea). qRT-PCR was carried out using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and the protocol in detail was previously described [18 (link)]. The expression of target genes was measured and normalized relative to the control house-keeping gene, 18S rRNA [38 (link),39 (link),40 (link)]. The gene expression values were calculated as previously described [18 (link)]. The list of primers is presented in Table 1.

Spleen tissue or isolated CD4⁺ T cells from uninfected and 7-, 14-, 21- and/or 28-day infected hamsters were collected and RNA was isolated using the Qiagen RNeasy Mini Kit (for concentrations larger than 1 μg) or Ambion RNAqueous-Micro Total RNA Isolation Kit (for concentrations less than 1 μg RNA). Total RNA was DNase treated with Life Technologies Turbo DNA-Free kit and reverse transcribed into cDNA according to manufacturer’s protocol (High-Capacity cDNA Reverse Transcription Kit, Life Technologies). Primer sequences were designed using Genscript Primer Design Tool and the National Center for Biotechnology Information (NCBI) Primer-BLAST, which provided wider selection parameters. The Ensembl genome database was used to map exons and introns according to the mouse genome, and each primer set was designed to span an intron on the hamster target gene. Primer fidelity was confirmed by analysis of dissociation curves. The target genes for which primers were designed are detailed in S1 Table. Gene expression was determined in total spleen tissue, baby hamster kidney cells (BHK fibroblast cell line) and CD4⁺ splenocytes by SYBR green PCR on ViiA 7 Real-Time PCR System. Data was analyzed using comparative Ct method relative to uninfected BHK controls or uninfected hamster controls and using the 18S ribosomal RNA gene as the normalizer.