Quantitative RT-PCR Analysis of Neuronal Genes

Total RNA was extracted from colon ME and M/SM tissues, and from CBT-labeled colon specific DRG neurons by using the Qiagen RNeasy kit (Qiagen, Valencia, CA). One microgram of total RNA was reverse-transcribed with the SuperScript III First-Strand Synthesis System (Invitrogen) for quantitative RT-PCR with the Applied Biosystems 7000 real-time PCR system (Foster City, CA) [22 , 25 (link), 27 (link), 39 (link), 50 ]. The assay IDs for TaqMan detection of rat NGF, Nav1.6, Nav1.7, Nav1.8, and Nav1.9 mRNAs are Rn01533872_m1, Rn00591020_m1, Rn00570506_m1, Rn00568393_m1, and Rn00570487_m1, respectively (Applied Biosystems). For relative quantification of mRNA expression, real-time qPCR was performed with 40 ng of cDNA for the target gene and for the endogenous control 18S rRNA (Part no. 4352930E, Applied Biosystems).

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Lin Y.M., Fu Y., Winston J., Radhakrishnan R., Sarna S.K., Huang L.Y, & Shi X.Z. (2017). Pathogenesis of abdominal pain in bowel obstruction: Role of mechanical stress-induced upregulation of nerve growth factor in gut smooth muscle cells. Pain, 158(4), 583-592.

Publication 2016

RNeasy kit SuperScript III First-Strand Synthesis System 7000 real-time PCR system

18s rrna Assay Cdna Colon Gene Mrna Neurons Rt pcr Synthesis Tissues

Corresponding Organization :

Other organizations : The University of Texas Medical Branch at Galveston

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Variable analysis

independent variables

Total RNA extracted from colon ME and M/SM tissues
Total RNA extracted from CBT-labeled colon specific DRG neurons

dependent variables

Quantitative expression of NGF, Nav1.6, Nav1.7, Nav1.8, and Nav1.9 mRNAs

control variables

18S rRNA as endogenous control for relative quantification of mRNA expression

controls

No positive or negative controls were explicitly mentioned.

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