At the end of week 8 of the experiment, one bird was randomly selected from each replicate cage (n = 8) and individual blood were taken aseptically from the wing vein after starvation for 10 h. Serum samples were then obtained by centrifugation of blood at 3,000 rpm for 10 min at 4°C and stored at −20°C. These birds were slaughtered rapidly after blood collection, a little patch of the liver from each bird was immediately removed into RNAStore solution (TIANGEN Biotech., Co. Ltd., Beijing, China) and stored at −80°C for RNA extraction. The remainder liver sample was collected for determination of hepatic lipids contents. Afterward, the intestinal tract of each bird was separated and the mid-point of ileal segment was then removed into RNAStore solution (TIANGEN Biotech., Co. Ltd., Beijing, China) and stored at −80°C for RNA extraction. Besides, digesta sample in the ileum was collected and quick-froze by liquid nitrogen, followed by storage at −80°C.
Rnastore solution
Manufactured by Tiangen Biotech
Sourced in China
RNAstore solution is a reagent designed for the stabilization and preservation of RNA samples. It effectively inhibits RNase activity, preventing RNA degradation during storage at ambient temperature.
Automatically generated - may contain errors
Lab products found in correlation
Glutaraldehyde solution, by Solarbio (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Tianscript first strand cdna synthesis kit, by Tiangen Biotech (1 mentions)
Superreal premix plus sybr green, by Tiangen Biotech (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab kit, by ZSGB-BIO (1 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Beyotime (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Nebnext ultra rna library preparation kit, by New England Biolabs (1 mentions)
Paraformaldehyde solution, by Sinopharm (1 mentions)
Whatman, by Cytiva (1 mentions)
9 protocols using rnastore solution
1
Liver and Ileal Gene Expression
2
Immunohistochemical Analysis of BMP-7, Gremlin, and p-Smad1/5/8
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Rabbit anti-human anti-BMP-7 antibody was obtained from Abcam, Cambridge, UK (cat. no. ab56023). Rabbit anti-human anti-gremlin antibody was purchased from Wuhan Boster Biological Technology, Ltd., Wuhan, China (cat. no. BA2287). Rabbit anti-human anti-p-Smad1/5/8 antibody was purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA (cat. no. sc-12353-R). The 3,3′-diaminobenzidine tetrahydrochloride (DAB) kit was obtained from ZSGB-Bio, Beijing, China. RNAstore solution, TIANScript first-strand cDNA synthesis kit, and SuperReal PreMix Plus SYBR Green were obtained from Tiangen Biotech Co., Ltd., Beijing, China. RIPA lysis buffer and phenylmethylsulfonyl fluoride (PMSF) were obtained from Beyotime Institute of Biotechnology, (Haimen, China).
3
RNA-Seq Analysis of Midbrain Tissue
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The midbrain tissue was separated on ice and stored in RNA store solution (TIANGEN, Beijing, China). The first total RNA was extracted, using NEBNext®UltraTM RNA library preparation kit to generate a sequencing library, and adding the index code to the attribute sequence of each sample. The library preparations were sequenced on the Illumina HiSeq platform. Use Galaxy’s genome analysis tool to process all sample data, and use the Salmon one-step quantitative method in the analysis tool to calculate the TPM (Transcripts Per Million) value of each set of data. We use the DESeq2 software package in the analysis tool to identify the differentially expressed genes between different groups; we use DAVID to analyze the differentially expressed genes. P value < 0.05 is considered significant. Then, RT-PCR was performed to validate the differential gene.
4
Transcriptomic Analysis of Murine Cardiac Samples
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RNA samples of murine heart tissue were collected from C57BL/6 mice treated with or without DOX and stored with RNAStore solution at 4 °C (#DP408-02, Tiangen Biotech Co., Ltd.). RNA isolation, quality control, library construction, and sequencing were performed by the Beijing Genomics Institute (www.genomics.org.cn , BGI) using the BGISEQ-500 platform. Cleaned reads were then mapped to the GRCm38.P6 reference genome with Bowtie2 (v2.2.5)17 (link). Transcript abundances were measured with RSEM (v1.2.8)18 . Further bioinformatics analyses were all accomplished in RStudio with R (v3.5.3) and GSEA (v4.0.3) with GSKB (v1.14.0)19 –21 . Low count genes were removed by a cut-off at 0.5829 FPKM. Differential expression analyses were performed with limma (R packages v3.38.3) by a cut-off at 1.44-fold-change and P value < 0.0522 (link). KEGG pathway enrichment was performed with clusterProfiler (v3.10.1)23 (link). Plots performed with ggplot2 (v3.2.1) and ggpubr (v0.2.4)24 ,25 . Heatmap of 50 up-regulated genes and 50 down-regulated genes was plotted with pheatmap (v1.0.12)26 . All codes are available from the corresponding author upon request.
5
Shrimp Disease Sampling and Analysis
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A total of 113 live M. rosenbergii samples (body length 12–14 cm) were collected from local farmed ponds in Gaoyou, Jiangsu Province at the time periods of 26 June, 17–25 September, and 18–19 October 2021. Additionally, the samples collected on 26 June 2021 exhibited disease signs such as abnormal swimming, empty intestine and shell softening. In the progress of sampling, the hepatopancreas, intestine, gill and gonad tissues of these prawns were sampled and cut into three parts: one part was fixed in 2.5% glutaraldehyde solution (Solarbio, Beijing, China) for transmission electron microscopic (TEM) examination; the second part was fixed in 4% paraformaldehyde solution (Sinopharm, Beijing, China) for ISH detection and histopathological analysis; the third part was chopped up and kept in RNAstore solution (Tiangen, Beijing, China) and 95% ethanol (Sinopharm, Beijing, China) for molecular pathogen identification.
6
CMNV Detection in Co-Inhabiting Sea Cucumbers
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In June 2018, we found that the farming P. vannamei collected from polyculture ponds of shrimp and sea cucumber was infected with CMNV. Considering the capacity of CMNV cross-species infection and the needs of further exploration of its host range, four sea cucumber individuals (length 7–8 cm, co-inhabiting with the P. vannamei in the same polyculture pond) (Figure 1 a,b) were randomly collected for CMNV detection. The collected sea cucumber individuals looked normal, but the body was not compacted enough (somewhat soft) compared with healthy individuals. These sea cucumbers were primarily examined after dissecting the body along the longitudinal axis, and the thinning intestinal tissue (Figure 1 c) was selected to be divided into three parts and preserved. One part was preserved in 4% paraformaldehyde solution in PBS (PFA-PBS) (Sinopharm, Beijing, China) for ISH detection and histopathological analysis. Another part was fixed in 2.5% glutaraldehyde solution (Solarbio, Beijing, China) for electron microscopic examinations. Residual intestinal tissues were preserved in RNAstore solution (Tiangen, Beijing, China) for molecular biological analysis.
7
Tissue Collection for ESCC Analysis
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In this study, tissue samples from 210 patients with ESCC and 60 patients with normal esophageal mucosa were collected from the First A liated Hospital of Bengbu Medical College from January 2014 to December 2015. The tissue samples of ESCC were obtained from surgical specimens. None of the patients with ESCC included in this study had received chemotherapy or radiotherapy before the surgery. Detailed clinicopathological data of the study participants are presented in Table 1. Additionally, fresh saline tissue samples of ESCC and adjacent normal tissues were collected from 10 patients between August 2019 and December 2019 from the First A liated Hospital of Bengbu Medical College. These samples were placed in liquid nitrogen immediately for later use in western blot analysis. For detection of mRNA levels, the fresh tissues were immersed in RNA store solution (TIANGEN, Beijing, China) in the ratio of 1:10 and stored in liquid nitrogen at 4°C overnight.
8
Tissue Collection for ESCC Analysis
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In this study, tissue samples from 210 patients with ESCC and 60 patients with normal esophageal mucosa were collected from the First A liated Hospital of Bengbu Medical College from January 2014 to December 2015. The tissue samples of ESCC were obtained from surgical specimens. None of the patients with ESCC included in this study had received chemotherapy or radiotherapy before the surgery. Detailed clinicopathological data of the study participants are presented in Table 1. Additionally, fresh saline tissue samples of ESCC and adjacent normal tissues were collected from 10 patients between August 2019 and December 2019 from the First A liated Hospital of Bengbu Medical College. These samples were placed in liquid nitrogen immediately for later use in western blot analysis. For detection of mRNA levels, the fresh tissues were immersed in RNA store solution (TIANGEN, Beijing, China) in the ratio of 1:10 and stored in liquid nitrogen at 4°C overnight.
9
Comprehensive Sampling and Analysis of L. polyactis
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In August 2018 and August 2019, bottom trawl samplings were conducted at 120 designated sites (south of 38 • 20 N, west of 120 • 30 E, Figure 1) in the Yellow Sea and northern East China Sea. Three to five individuals of L. polyactis (Figure 2A) were sampled at every designated sampling site. From each individual, the tissues of liver, kidney, spleen, brain, heart, and ovary (if available) were dissected, and half of the tissues were preserved in 4% paraformaldehyde (4% PFA) solution (Sinopharm, Beijing, China) for histopathological and in situ hybridization analysis. Another half was further divided into two parts, one part was preserved in RNAstore solution (Tiangen, Beijing, China) for RT-PCR analyses, the other part was smeared on Whatman FTA R Elute card (GE Healthcare Life Sciences, Marlborough, MA, United States) for RT-LAMP analyses. The tissue-smeared FTA R cards were air dried for 30 min at room temperature, and then stored at -20 • C.
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