The developmental stage-specific expression profiles of B. dorsalis were established using samples of eggs, larvae (1-, 4-, 7-day-old), pupae (1-, 4-, 7-day-old), and adults (1-, 5-, 9-day-old). Five randomly collected insects were pooled as one sample for stage-specific expression profiling, with three independent biological replications per stage. Tissue from the CNS (both brain and ventral nerve cord), gut (the complete digestive tract), fat body, Malpighian tubules, epidermis, and the epitracheal gland (EG), containing the Inka cells, were excised from 2-day-old 3rd-instar larvae (i.e., 7-day-old larvae; this is the moment prior to larval-pupal transition) to determine the tissue-specific expression patterns. At least 15 individuals were dissected as one sample for tissue-specific analysis, and three independent biological replications were done per tissue. The larvae were chilled on ice for 30 min and dissected under a stereomicroscope (Olympus SZX12, Tokyo, Japan). The samples were isolated on ice, placed in a 2.0 mL-diethyl pyrocarbonate-treated centrifuge tube containing RNA storage reagent (Tiangen, Beijing, China), and immediately frozen in liquid nitrogen and stored at −80°C.
Rna storage reagent
Manufactured by Tiangen Biotech
Sourced in China
The RNA storage reagent is a solution designed to preserve and stabilize RNA samples. It prevents degradation of RNA molecules, ensuring their integrity for downstream applications.
Automatically generated - may contain errors
4 protocols using rna storage reagent
1
Developmental and Tissue-Specific Expression Profiles in Bactrocera dorsalis
2
Multistage Insect Development and Tissue-Specific Sampling
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The laboratory stock colony of L. serricorne, originally collected in 2014 from a tobacco warehouse in Guizhou Province, China, was reared on Chinese medicinal material (Angelica sinensis) and maintained at 28 ± 1 °C and 40% ± 5% relative humidity under a scotoperiod of 24 h.
Samples at different developmental stages, including early larvae (EL, <24 h post-hatching), late larvae (LL, older than fourth instar larvae and before prepupae), pupae (PU, >48 h post-pupation), early adults (EA, <24 h post-eclosion), and late adults (LA, one week old) were collected separately and stored at −80 °C. In the tissue-specific experiment, the fifth instar larvae were used for tissue isolation. The integument, fat body, gut, and carcass of L. serricorne were dissected under a stereomicroscope (Olympus SZX12, Tokyo, Japan). Each tissue type was placed in a 1.5-mL centrifuge tube containing RNA storage reagent (Tiangen, Beijing, China). Pools of 30 individuals of larvae were used to prepare the integument, gut, and carcass, and 50 individuals were pooled to collect the fat body tissue. All tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C. Each sample was replicated three times.
Samples at different developmental stages, including early larvae (EL, <24 h post-hatching), late larvae (LL, older than fourth instar larvae and before prepupae), pupae (PU, >48 h post-pupation), early adults (EA, <24 h post-eclosion), and late adults (LA, one week old) were collected separately and stored at −80 °C. In the tissue-specific experiment, the fifth instar larvae were used for tissue isolation. The integument, fat body, gut, and carcass of L. serricorne were dissected under a stereomicroscope (Olympus SZX12, Tokyo, Japan). Each tissue type was placed in a 1.5-mL centrifuge tube containing RNA storage reagent (Tiangen, Beijing, China). Pools of 30 individuals of larvae were used to prepare the integument, gut, and carcass, and 50 individuals were pooled to collect the fat body tissue. All tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C. Each sample was replicated three times.
3
Tissue-Specific Expression Analysis
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Tissues from the CNS, fat body, midgut, hindgut, Malpighian tubules, and testis were excised from 9-day-old adults to determine the tissue-specific expression pattern. At least 15 individuals were dissected as one sample for tissue-specific analysis with four replications. The adults were chilled on ice for 30 min and dissected under a stereomicroscope (Olympus SZX12, Tokyo, Japan). The samples were isolated on ice, placed in a 2.0 mL of diethyl pyrocarbonate (DEPC)-treated centrifuge tube containing RNA storage reagent (Tiangen, Beijing, China), and immediately frozen in liquid nitrogen and stored at -80°C.
Total RNA was extracted from each sample using the TRIzol reagent (Invitrogen, Carlsbad, CA, United States) and treated with DNase I (Promega, Madison, WI, United States) to prevent potential genomic DNA contamination. First-strand cDNA was synthesized using the GoScript Reverse Transcription System (Promega) according to the manufacturer’s instructions.
Total RNA was extracted from each sample using the TRIzol reagent (Invitrogen, Carlsbad, CA, United States) and treated with DNase I (Promega, Madison, WI, United States) to prevent potential genomic DNA contamination. First-strand cDNA was synthesized using the GoScript Reverse Transcription System (Promega) according to the manufacturer’s instructions.
4
RNA-seq Analysis of Mammary Tumors
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Nine tumor samples from the FVB/N-Tg(MMTV-PyMT)634Mul and Fvb.B6 F1 hybrids were placed in RNA storage reagent (TIANGEN) for RNA isolation. Total RNA was isolated from the 18 samples, with three tumor samples per group. RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). The RNA was sheared and reverse transcribed using random primers to obtain the cDNA for library construction. The library was sequenced through the BGISEQ-500 platform at the Beijing Genomic Institution (Wuhan, China).
All statistical analyses were performed by using R v3.3.1. After removing transcriptionally inactive genes (read counts per million were < 1), the high-confidence gene counts were obtained. For gene expression analysis, the matched reads were calculated and normalized to RPKM using RESM software [19 (link)]. The R package edgeR v3.18.1 was used to perform statistical analyses on the gene counts and to detect differentially expressed genes (DEGs). DEGs at each stage were used for further analyses of GO (gene ontology), biological processes and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways using the R package clusterProfiler v3.4.4 [20 ]. The heatmap of the immune-related differentially expressed genes was constructed using the R package pheatmap v1.0.8.
All statistical analyses were performed by using R v3.3.1. After removing transcriptionally inactive genes (read counts per million were < 1), the high-confidence gene counts were obtained. For gene expression analysis, the matched reads were calculated and normalized to RPKM using RESM software [19 (link)]. The R package edgeR v3.18.1 was used to perform statistical analyses on the gene counts and to detect differentially expressed genes (DEGs). DEGs at each stage were used for further analyses of GO (gene ontology), biological processes and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways using the R package clusterProfiler v3.4.4 [20 ]. The heatmap of the immune-related differentially expressed genes was constructed using the R package pheatmap v1.0.8.
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