LCMV RNA Quantification from Spleen Samples

As previously described (3 (link), 18 (link)) small (~5mg) pieces of spleen were harvested and stored in RNA later (Qiagen). RNA was isolated using RNeasy mini (Qiagen) with on-column DNA digestion according to manufacturer instructions. cDNA was generated using the primer 5′-CAGGGTGCAAGTGGTGTGGTAAGA-3′ (Valuegene, CA), specific for the genomic (negative) sense of the LCMV nucleoprotein RNA, at 200nM with Multiscribe-RT (Applied Biosystems, Ca). TaqMan real time PCR was performed with the following primers (Valuegene) and probe (Applied Biosystems): forward primer (900nM) 5′-CGCTGGCCTGGGTGAAT-3′, reverse primer (900nM) 5′-ATGGGAAAACACAACAATTGATCTC-3′ and probe (200nM) 6FAM-CTGCAGGTTTCTCGC-MGBNFQ. LCMV genome copy number was calculated from standard dilutions of the plasmid pT7-S which encodes the S segment of LCMV (19 (link)) (a kind gift of Professor Juan Carlos de la Torre, TSRI). Data were acquired on a BioRad iCycler and analyzed with iCycler iQ software (BioRad, CA).

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Hosking M.P., Flynn C.T, & Whitton J.L. (2014). Antigen-specific naïve CD8+ T cells produce a single pulse of IFNγ in vivo within hours of infection, but without antiviral effect. Journal of immunology (Baltimore, Md. : 1950), 193(4), 1873-1885.

Publication 2014

RNA later RNeasy mini Multiscribe-RT iCycler iCycler iQ software

Cdna Digestion Dilutions Genome Lcmv Nucleoprotein Plasmid Primer Real time pcr Spleen

Corresponding Organization : Scripps Research Institute

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Variable analysis

independent variables

Quantity of spleen pieces harvested (3 or 18 pieces)

dependent variables

LCMV genome copy number

control variables

RNA isolation using RNeasy mini (Qiagen) with on-column DNA digestion
CDNA generation using the primer 5′-CAGGGTGCAAGTGGTGTGGTAAGA-3′ (Valuegene, CA)
TaqMan real-time PCR with the specified primers and probe
Data acquisition on a BioRad iCycler and analysis with iCycler iQ software (BioRad, CA)

controls

Positive control: Plasmid pT7-S which encodes the S segment of LCMV (a kind gift of Professor Juan Carlos de la Torre, TSRI)
Negative control: Not explicitly mentioned

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