Computational protocol: Critical evaluation of the expression of gastrin-releasing peptide in dorsal root ganglia and spinal cord

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Protocol publication

[…] RT-PCR was performed as previously described. Mice were sacrificed. DRG and spinal cord tissues were quickly dissected and rapidly frozen in dry ice. Total RNA was isolated and genomic DNA was removed in accordance with manufacturer’s instructions (RNeasy plus mini kit; QIAGEN). Single-stranded cDNA (total of 20 µL per sample) was synthesized from 1 µg RNA by using High Capacity cDNA Reverse Transcription Kit (Life Technologies). Gene expression of Grp and Tac1 was determined by real-time PCR (StepOnePlus; Applied Biosystems). Specific intron-spanning primers were designed with the NCBI Primer-BLAST. The primers used are:Actb (NM_007393.3): 5’-TGTTACCAACTGGGACGACA-3’;5’-GGGGTGTTGAAGGTCTCAAA-3’; amplicon size: 166 bp.Gapdh (NM_008084.2): 5’-CCCAGCAAGGACACTGAGCAA-3’;5’-TTATGGGGGTCTGGGATGGAAA-3’; amplicon size: 93 bp.Grp (NM_175012.3): 5’-TGGGCTGTGGGACACTTAAT-3’; 5’-GCTTCTAGGAGGTCCAGCAAA-3’; amplicon size: 146 bp.Tac1 (NM_009311.2): 5’-GTGGCCCTGTTAAAGGCTCT-3’; 5’-TGCCCATTAGTCCAACAAAGGA-3’; amplicon size: 85 bp.Actb (NM_007393.3): 5’-TGTTACCAACTGGGACGACA-3’;5’-GGGGTGTTGAAGGTCTCAAA-3’; amplicon size: 166 bp.Gapdh (NM_008084.2): 5’-CCCAGCAAGGACACTGAGCAA-3’;5’-TTATGGGGGTCTGGGATGGAAA-3’; amplicon size: 93 bp.Grp (NM_175012.3): 5’-TGGGCTGTGGGACACTTAAT-3’; 5’-GCTTCTAGGAGGTCCAGCAAA-3’; amplicon size: 146 bp.Tac1 (NM_009311.2): 5’-GTGGCCCTGTTAAAGGCTCT-3’; 5’-TGCCCATTAGTCCAACAAAGGA-3’; amplicon size: 85 bp.Real-time PCR was carried out with FastStart Universal SYBR Green Master (Roche Applied Science). All samples were assayed in duplicates. PCR (heating at 95℃ for 10 s and 60℃ for 30 s) were performed. Data were analyzed using Comparative CT Method (StepOne Software v2.2.2.), and the expression of target mRNA was normalized to the expression of Actb and Gapdh.RT-PCR with 2% agarose gel electrophoresis was performed using Taq DNA Polymerase (New England Biolabs) with 2 µL cDNA as a template per reaction. Reactions were optimized by running annealing temperature and cycle gradients with no-RT (ΔRT) and no-template (H2O) samples as negative controls. The following intron-spanning primer pairs and PCR parameters were used:Actb: 5’-GATGACGATATCGCTGCGCTGGTCG-3’;5’-GCCTGTGGTACGACCAGAGGCATACA-3’; amplicon size 447 bp. Parameters: 95℃ 5 min, [95℃ 40 s, 55℃ 40 s, 72℃ 40 s] for 21 cycles, 72℃ 10 min.Grp: 5’-AGTCGAGAGCTCTGAGGGTT-3’; 5’-CCCTTGTCGTTGTCCCTTCA-3’; amplicon size 491 bp. Parameters: 95℃ 5 min, [95℃ 40 s, 61.4℃ 40 s, 72℃ 40 s] for 35 cycles, 72℃ 10 min.Nppb (): 5’-CTTTATCTGTCACCGCTGGG-3’;5’-AGGAGGTCTTCCTACAACAACTTC-3’; amplicon size 329 bp. Parameters: 95℃ 5 min, [95℃ 40 s, 55℃ 40 s, 72℃ 40 s] for 28 cycles, 72℃ 10 min.Tac1: 5’-GAGAGCAAAGAGCGCCCAG-3’; 5’-AAGAGCCTTTAACAGGGC-3’; amplicon size 329 bp. Parameters: 95℃ 5 min, [95℃ 40 s, 55℃ 40 s, 72℃ 40 s] for 24 cycles, 72℃ 10 min.Actb: 5’-GATGACGATATCGCTGCGCTGGTCG-3’;5’-GCCTGTGGTACGACCAGAGGCATACA-3’; amplicon size 447 bp. Parameters: 95℃ 5 min, [95℃ 40 s, 55℃ 40 s, 72℃ 40 s] for 21 cycles, 72℃ 10 min.Grp: 5’-AGTCGAGAGCTCTGAGGGTT-3’; 5’-CCCTTGTCGTTGTCCCTTCA-3’; amplicon size 491 bp. Parameters: 95℃ 5 min, [95℃ 40 s, 61.4℃ 40 s, 72℃ 40 s] for 35 cycles, 72℃ 10 min.Nppb (): 5’-CTTTATCTGTCACCGCTGGG-3’;5’-AGGAGGTCTTCCTACAACAACTTC-3’; amplicon size 329 bp. Parameters: 95℃ 5 min, [95℃ 40 s, 55℃ 40 s, 72℃ 40 s] for 28 cycles, 72℃ 10 min.Tac1: 5’-GAGAGCAAAGAGCGCCCAG-3’; 5’-AAGAGCCTTTAACAGGGC-3’; amplicon size 329 bp. Parameters: 95℃ 5 min, [95℃ 40 s, 55℃ 40 s, 72℃ 40 s] for 24 cycles, 72℃ 10 min. [...] RNA-seq reads were aligned to the GRCm38.76 assembly from Ensembl with STAR version 2.0.4 b. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.5. Transcript counts were produced by Sailfish version 0.6.3. Sequencing performance was assessed for total number of aligned reads, total number of uniquely aligned reads, genes and transcripts detected, ribosomal fraction known junction saturation and read distribution over known gene models with RSeQC version 2.3. All gene-level and transcript counts were then imported into the R/Bioconductor package EdgeR and TMM normalized to adjust for differences in library size. Genes or transcripts not expressed in any sample were excluded from further analysis. Performance of the samples was assessed with a spearman correlation matrix and multi-dimensional scaling plots. Generalized linear models with robust dispersion estimates were created to test for gene/transcript level differential expression. The fit of the trended and tagwise dispersion estimates were then plotted to confirm proper fit of the observed mean to variance relationship where the tagwise dispersions are equivalent to the biological coefficients of variation of each gene. Differentially expressed genes and transcripts were then filtered for FDR adjusted p values less than or equal to 0.05.To enhance the biological interpretation of the large set of transcripts, grouping of genes/transcripts based on functional similarity was achieved using the R/Bioconductor packages GAGE and Pathview. GAGE and Pathview were also used to perform pathway maps on known signaling and metabolism pathways curated by KEGG. […]

Pipeline specifications

Software tools Primer-BLAST, STAR, Subread, RSeQC, edgeR
Applications RNA-seq analysis, qPCR
Organisms Mus musculus
Chemicals Substance P