Tick Gene Expression Analysis

Ticks were allowed to feed for 24 h, for 72 h, or to repletion (between 80 and 96 h after initiation of tick feeding) and RNA isolated from guts and salivary glands using Trizol (Invitrogen, CA) as described earlier [22] (link). cDNA was synthesized using the iScript RT-PCR kit (Bio-Rad, CA) and analyzed by quantitative PCR for the expression of tick actin and B. burgdorferi and also ixofin3D, clone 2, 3 and 4 using gene-specific primers (listed in table S1) and the iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) on a Opticon Engine MJ cycler (Bio-Rad, CA).

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Narasimhan S., Coumou J., Schuijt T.J., Boder E., Hovius J.W, & Fikrig E. (2014). A Tick Gut Protein with Fibronectin III Domains Aids Borrelia burgdorferi Congregation to the Gut during Transmission. PLoS Pathogens, 10(8), e1004278.

Publication 2014

Trizol iScript RT-PCR kit iQ SYBR Green Supermix

Actin Cdna Clone Gene Guts Primers Rt pcr Salivary glands Sybr green Tick Trizol

Corresponding Organization : Howard Hughes Medical Institute

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Variable analysis

independent variables

Duration of tick feeding (24 h, 72 h, or to repletion between 80 and 96 h)

dependent variables

Expression of tick actin
Expression of B. burgdorferi
Expression of ixofin3D, clone 2, 3 and 4

control variables

Tick guts and salivary glands used for RNA isolation
Use of Trizol (Invitrogen, CA) for RNA isolation
Use of iScript RT-PCR kit (Bio-Rad, CA) for cDNA synthesis
Use of gene-specific primers (listed in table S1) and iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) for quantitative PCR analysis
Use of Opticon Engine MJ cycler (Bio-Rad, CA) for quantitative PCR

controls

No positive or negative controls are explicitly mentioned in the provided information.

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